HUMAN FIBROLAMELLAR HEPATOCELLULAR CARCINOMAS (hFL-HCCS)

ABSTRACT

The present disclosure provides a model of human fibrolamellar hepatocellular carcinoma (FL-HCC) cells maintained as a transplantable tumor line in a host and a method to establish a transplantable human FL-HCC tumor line. Methods of ex vivo cultures of the FL-HCC are provided. Methods of diagnosing and treating FL-HCC tumors are also provided.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application claims priority from U.S. Provisional Application62/129,668, filed Mar. 6, 2015, incorporated herein by reference in itsentirety.

GOVERNMENT SUPPORT

This invention was made with government support under federal NIH grantROODK091318-02, awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND

Human fibrolamellar hepatocellular carcinomas (hFL-HCCs) are rarecancers accounting for less than ˜5% of all liver cancers and unique inbeing found primarily in children to young adults without evidence offibrosis or cirrhosis. The epidemiological factors are unknown, as arecauses of increases in occurrence in hFL-HCCs over the past 60 years.These malignances are currently treatable only by surgery, as all testedforms of chemotherapy and external radiation therapy have provenineffective. Even surgery is ineffective if the hFL-HCC tumor hasmetastasized. In addition, molecular mechanisms of hFL-HCCs have beendifficult to study, since investigations have had to be conducted onfreshly isolated tissue or paraffin sections-samples that are difficultto obtain. Therefore, a need exists for in vivo models of hFL-HCCs, suchas transplantable tumor lines, and/or in vitro models of hFL-HCCs, suchas cell lines or spheroid cultures, for use in defining the disease aswell as identifying novel strategies for treating hFL-HCCs.

SUMMARY

Aspects of the disclosure relate to transplantable tumor lines of humanfibrolamellar hepatocellular carcinoma (hFL-HCC) cells maintained in anon-human animal. In some aspects, the transplantable tumor linecomprises hFL-HCC cells and mesenchymal cells from a non-human host.Also disclosed is a composition comprising hFL-HCC cells and an amountof non-human mesenchymal cells effective to sustain the viability ofsaid hFL-HCC cells.

Other aspects of the disclosure provide cell cultures comprising hFL-HCCcells in a serum-free medium.

Further aspects provide methods for establishing a hFL-HCC tumor linecomprising: (a) obtaining a hFL-HCC tumor from a patient with a FL-HCC;(b) preparing a tumor cell suspension from the FL-HCC tumor; (c)culturing the tumor cell suspension under restrictive conditions thatselect for cancer stem cells to obtain a population of culture-selectedcancer stem cells; and (d) transplanting culture-selected cells into animmunocompromised, non-human animal.

Aspects of the disclosure also relate to methods for maintaining ahFL-HCC transplantable tumor line comprising: (a) obtaining hFL-HCCcells from a xenografted tumor of a first immunocompromised non-humananimal; (b) dispersing the hFL-HCC cells into a cell suspension byenzymatic or mechanical methods; and (c) transplanting dispersed hFL-HCCcells into a second immunocompromised, non-human animal.

Additional aspects provide methods for culturing hFL-HCC cellscomprising: (a) separating hFL-HCC cells of a xenografted tumor fromnon-human cells; (b) suspending the separated hFL-HCC cells in aserum-free medium; and (c) plating the hFL-HCC cells onto or into aculture substratum to obtain plated hFL-HCC cells.

Additional aspects provide methods for culturing hFL-HCC cellscomprising: (a) separating hFL-HCC cells of a xenografted tumor fromnon-human cells; (b) suspending the separated hFL-HCC cells in aserum-free medium; and (c) allowing the cells to form floatingaggregates (e.g. spheroids or organoids) in a culture medium.

In some aspects herein provided are methods for drug screening,comprising (a) introducing a candidate drug to cultured hFL-HCC cellsthat are in the form of monolayers, hydrogels, spheroids, or organoidsand (b) monitoring the effect of the candidate drug on the culturedhFL-HCC cells.

In some aspects herein provided are methods for drug testing, comprising(a) administering a candidate drug to a non-human animal carrying atransplantable hFL-HCC tumor and (b) monitoring the effect of thecandidate drug on the xenotransplanted hFL-HCC tumor.

In some aspects herein provided are methods for suppressing the growthof hFL-HCC cells, comprising treating the hFL-HCC cells with a drug, animmunotherapy, or an inhibitor to a specific signaling pathway.Non-limiting examples include a hedgehog signaling pathway inhibitor, ahistone deacetylase inhibitor and/or an inhibitor to one or more proteinkinases. Some specific examples include, inhibitor of CA12 such asAcetazolamide, and/or anti-sense oligonucleotides to SLC16A14 tominimize its effects conferring drug resistance, which is relevant tohFL-HCC, a cancer that is highly chemo- and drug-resistant.

Further aspects provide methods for treating hFL-HCC in a patient inneed thereof, comprising administering to the patient an effectiveamount of drug, with immunotherapy, with an inhibitor to a specificsignaling pathway. Non-limiting examples include a hedgehog signalingpathway inhibitor, a histone deacetylase inhibitor and/or an inhibitorto one or more protein kinases.

In yet other aspects herein provided are methods of determining whethera patient has fibrolamellar hepatocellular carcinoma (FL-HCC),comprising: (a) measuring gene expression levels of at least one ofC10orf128, CA12, CREB3L1, GALNTL6, IRF4, ITPRIP, KCNE4, NOVA1, OAT,PAK3, PCSK1, PHACTR2, RPS6KA2, SLC16A14, TMEM163, and TNRC6C; and (b)comparing the gene expression profile to one or more control samples.

In yet other aspects herein provided are methods of determining whethera patient has fibrolamellar hepatocellular carcinoma (FL-HCC),comprising: (a) measuring gene expression levels of at least one or moregenes of a set of genes found to constitute a genetic signature forhFL-HCCs and that include C10orf128, CA12, CREB3L1, GALNTL6, IRF4,ITPRIP, KCNE4, NOVA1, OAT, PAK3, PCSK1, PHACTR2, RPS6KA2, SLC16A14,TMEM163, and TNRC6C from a sample collected from a patient suspected ofhaving a biliary tree or liver tumor; and (d) comparing the geneexpression profile to one or more control samples, wherein the samplecollected from the patient has histological features typical for FL-HCCand/or expresses the DNAJB1-PRKACA fusion gene.

In yet other aspects herein provided are methods of treating a patientdetermined to have hFL-HCC by administering to the patient an effectiveamount of at least one therapeutic that decreases expression of at leastone gene in a set found to be a genetic signature for hFL-HCCs and thatinclude C10orf128, CA12, CREB3L1, GALNTL6, IRF4, ITPRIP, KCNE4, NOVA1,OAT, PAK3, PCSK1, PHACTR2, RPS6KA2, SLC16A14, TMEM163, or TNRC6C.

In yet other aspects herein provided are methods of treating a patientdetermined to have hFL-HCC by administering to the patient an effectiveamount of an immunotherapy.

In yet other aspects herein provided are methods of treating a patientdetermined to have hFL-HCC by administering to the patient an effectiveamount of at least one therapeutic that regulates PRKACA or SRC networkhubs.

In yet other aspects herein provided are methods of treating a patientdetermined to have hFL-HCC by administering to the patient an effectiveamount of at least one therapeutic that regulates substrate targets ofthe kinase PRKACA (Protein kinase A catalytic subunit alpha).

In another aspect are provided compositions of isolated hFL-HCC cellswherein the hFL-HCC cell expresses at least one marker selected from thegroup consisting of C10orf128, CA12, CREB3L1, GALNTL6, IRF4, ITPRIP,KCNE4, NOVA1, OAT, PAK3, PCSK1, PHACTR2, RPS6KA2, SLC16A14, TMEM163, andTNRC6C.

In yet other aspects, are provided herein, populations of isolatedhFL-HCC cells wherein the hFL-HCC cell expresses a marker selected fromthe group consisting of C10orf128, CA12, CREB3L1, GALNTL6, IRF4, ITPRIP,KCNE4, NOVA1, OAT, PAK3, PCSK1, PHACTR2, RPS6KA2, SLC16A14, TMEM163, andTNRC6C.

In still yet other aspects provided herein are compositions comprisingisolated hFL-HCC cells wherein the hFL-HCC cell expresses a markerselected from the group consisting of C10orf128, CA12, CREB3L1, GALNTL6,IRF4, ITPRIP, KCNE4, NOVA1, OAT, PAK3, PCSK1, PHACTR2, RPS6KA2,SLC16A14, TMEM163, and TNRC6C and a carrier.

One embodiment of the disclosure described herein relates to a specifictransplantable human tumor line, TU-2010, consisting of hFL-HCC tumorcells and large numbers (>50% of the cells in the tumor) of mesenchymalcells of the non-human host.

In some embodiments, the non-human animal is immunocompromised. In someembodiments, the non-human animal is a mouse, for example, a NOD scidgamma (NSG) mouse.

In some embodiments, the hFL-HCC cells are derived from liver, frombiliary tree, from a subcutaneous or intraperitoneal tumor, for example,ascites tumor cells.

In some embodiments, the tumor line comprises hFL-HCC cells andmesenchymal cells of the non-human animal. In some embodiments, at least50% of the hFL-HCC cells in the transplantable tumor are cancer stemcells.

In some embodiments, the hFL-HCC cells express the fusion transcriptDNAJB1-PRKACA. In other embodiments, the hFL-HCC cells substantially donot express HDAC9 or express a lower level of HDAC9 as compared to ahuman non-FL-HCC cell control sample.

In some embodiments, the hFL-HCC cells express one or more markers ofendodermal transcription factors selected from the group consisting ofSOX9, SOX17, PDX1, FOXA1, and NGN3.

In some embodiments, the hFL-HCC cells express one or more markers ofpluripotency genes selected from the group consisting of OCT4, SOX2,NANOG, KLF4, SALL4 and KLF5.

In some embodiments, the hFL-HCC cells express one or more markers ofother stem cell genes selected from the group consisting of CD44, SALL4,TROP-2, BMI-1, sonic hedgehog (SHH), LGR5, NCAM, and KRT20.

In some embodiments, the hFL-HCC cells express one or more hepaticmarkers selected from the group consisting of CK8, CK18, CK19, DCLK1,HepPar-1, albumin, alpha-fetoprotein, and CD68.

In some embodiments, the hFL-HCC cells express one or more pancreaticmarkers selected from PDX1, NGN3, PCSK1, insulin, glucagon, amylase, andmucin (MUC).

In some embodiments, the hFL-HCC cells express high levels of arylhydrocarbon receptors (AHR).

In some embodiments, the hFL-HCC cells express biomarkers of malignancysuch as AGR2 and/or high levels of extracellular matrix-degradingenzymes and/or aberrations in the regulation of p53.

In some embodiments, the hFL-HCC cells have aberrant or lack ofexpression of one or more histone deacetylase (HDAC) genes.

In some embodiments, the tumor is a xenotransplanted, subcutaneous orintraperitoneal tumor.

In some embodiments, at least 30% of the hFL-HCC cells are cancer stemcells (CSCs). In other embodiments, at least 50% of the hFL-HCC cellsare CSCs. In yet other embodiments, at least 65% of the hFL-HCC cellsare CSCs. In still other embodiments, at least 51% of the cells in thecell culture are hFL-HCC cells.

In some embodiments, provided herein is a tissue sample obtained fromthe tumor line of any one of the above embodiments.

In some embodiments, the serum-free medium is Kubota's Medium. In someembodiments, the serum-free medium contains hyaluronans, HGF and/orVEGF.

In some embodiments, at least a portion of the hFL-HCC cells are inaggregates (e.g. spheroids) of hFL-HCC cells. In other embodiments, atleast a portion of the hFL-HCC cells are in organoids comprised ofhFL-HCCs and associated mesenchymal cells (e.g. endothelia, stellatecells, stromal cells).

In some embodiments, the hFL-HCC tumor is obtained as an ascites fluidor as a solid tumor from the subject.

In some embodiments, the tumor cell suspension from the hFL-HCC tumorare cultured on tissue culture plastic, on hyaluronans, or in hyaluronanhydrogels.

In some embodiments, the tumor cell suspension from the hFL-HCC tumorare cultured in serum-free Kubota's Medium.

In some embodiments, the methods provided herein comprise transplantingsubcutaneously or intraperitoneally the culture-selected cancer stemcells from the hFL-HCC cells into the immunocompromised non-humananimal.

In some embodiments, the methods provided herein comprise transplantingabout 10² to about 10⁷ culture-selected cancer stem cells from thehFL-HCC tumor into the immunocompromised, non-human animal.

In some embodiments, the methods provided herein further comprisemonitoring the immunocompromised, non-human animal for tumor formationfor about 2 to about 9 months

In some embodiments, the methods provided herein further comprisetransplanting subcutaneously or intraperitoneally the hFL-HCC tumor intothe second immunocompromised, non-human animal.

In some embodiments, the methods provided herein comprise separatinghFL-HCC cells from non-human cells by immunoselection, for example,magnetic immunoselection.

In some embodiments, the culture substratum is tissue culture plastic, a2D monolayer or 3D hydrogel of a purified extracellular matrixcomponent. In some embodiments, the purified extracellular matrixcomponent is selected from the group consisting of hyaluronan, acollagen, an adhesion molecule, and an extract enriched in extracellularmatrix. In some embodiments, the adhesion molecule is laminin. In otherembodiments, the extract enriched in extracellular matrix is a biomatrixscaffold or Matrigel. In some embodiments, the matrix scaffold isprepared by protocols for decellularized tissue. In other embodiments,the matrix scaffold is prepared from high salt decellularizationprotocols, such as biomatrix scaffolds.

In some embodiments, the plated hFL-HCC cells are kept in suspension andallowed to form aggregates, for example, spheroids (only the hFL-HCCcells) or organoids (mixtures of hFL-HCC cells and mesenchymal cells(endothelia, stellate cells, stromal cells).

In some embodiments, the hedgehog signaling pathway inhibitor comprisesGDC-0449.

In some embodiments, the histone deacetylase inhibitor comprisessuberoylanilide hydroxamic acid (SAHA) and/or suberic bis-hydroxamicacid (SBHA).

In some embodiments, overexpression of C10orf128, CA12, CREB3L1,GALNTL6, IRF4, ITPRIP, KCNE4, NOVA1, OAT, PAK3, PCSK1, PHACTR2, RPS6KA2,SLC16A14, TMEM163 or TNRC6C relative to the control sample is associatedwith presence of hFL-HCC. In some embodiments, overexpression of PCSK1,CA12, NOVA1, SLC16A14, TNRC6C, TMEM163, and RPS6KA2 relative to thecontrol sample is associated with presence of hFL-HCC. In otherembodiments, overexpression of C10orf128, OAT, PAK3, PCSK1, PHACTR2,SLC16A14, TMEM163, and TNRC6C relative to the control sample isassociated with presence of hFL-HCC. In some embodiments, the hFL-HCCalso expresses the fusion gene DNAJB1-PRKACA.

In some embodiments, the control sample is selected from the groupconsisting of hepatocellular carcinomas (HCCs), hepatoblastomas,cholangiocarcinomas (CCAs), pancreatic cancers, other types of cancersas well as normal cells that include biliary tree stem cells, hepaticstem cells, hepatoblasts, pancreatic stem cells, hepatic or pancreaticcommitted progenitors, and mature liver or pancreatic cells.

In some embodiments, the at least one therapeutic is selected from thegroup consisting of a small molecule, RNA interference, and a lockednucleic acid (LNA). Alternatively, at least one therapeutic is selectedfrom a form of immunotherapy.

These and other features, together with the organization and manner ofoperation thereof, will become apparent from the following detaileddescription when taken in conjunction with the accompanying drawings.

ABBREVIATIONS AND TERMINOLOGY

Acronyms for cell populations are preceded by a small letter to indicatethe species: m=murine; h=human. ABCG2 (or CDw338), ATP-binding cassettesub-family G member 2 that confers drug resistance; Acetazolamide, aninhibitor of carbonic anhydrases; AFP, α-fetoprotein; ALB, albumin;Basal Media, buffers comprised of amino acids, minerals, sugars, lipids,vitamins and other nutrients in a composition mimicking interstitialfluid and used for cell culture; BMi-1, B lymphoma Mo-MLV insertionregion 1 homolog that is an oncogene conferring the ability ofself-replication of cells; BTSCs, biliary tree stem cells; CAJ2,carbonic anhydrase 12, a zinc metallo enzyme; Clorf128, Chromosome 10open reading frame 128; CCA, cholangiocarcinoma; CD, common determinant;CD13, alanine aminopeptidase; CD44, hyaluronan receptors; CD133,prominin; CFTR, cystic fibrosis transmembrane conductance regulator; CK,cytokeratin protein; CREB3L1, the cAMP responsive element bindingprotein 3-like 1; CSCs, cancer stem cells; CXCR4, CXC-chemokine receptor4 (also called fusin or CD184); EGF, epidermal growth factor; EpCAM,epithelial cell adhesion molecule; FBS, fetal bovine serum; FGF,fibroblast growth factor; FLC, fibrolamellar carcinoma (synonym=FL-HCC,fibrolamellar hepatocellular carcinoma); GALNT6, polypeptideN-acetyl-galactos-aminyl-transferase-like 6 that participates in0-glycan biosynthesis; GDC-0449, inhibitor of hedgehog signaling pathwayvia hedgehog surface receptors (PTCH, SMO); HDAC, histone deacetylase;HDM, a serum-free medium comprised of basal media and a defined mix ofpurified hormones, growth factors and nutrients tailored for a specificcell or biological process; HDM-C, a hormonally defined medium forcholangiocytes; HDM-H, a hormonally defined medium for hepatocytes;HDM-P, a hormonally defined medium for pancreatic islets; hFL-HCC, humanfibrolamellar hepatocellular carcinoma; HBs, hepatoblasts; HCC,hepatocellular carcinoma; HGF, hepatocyte growth factor; HpSCs, hepaticstem cells; IRF4, Interferon regulatory factor 4. Transcriptionalactivator; ITPRIP, inositol 1,4,5-trisphosphate receptor-interactingprotein; KCNE4: Potassium voltage-gated channel subfamily E, member 4modulates the multimeric channel complex. KM, Kubota's Medium, aserum-free, hormonally defined medium designed for endodermalstem/progenitors; KRT, cytokeratin gene; LGR5, Leucine-richrepeat-containing G-protein coupled receptor 5 that binds to R-spondin;NANOG, a transcription factor critically involved with self-renewal;NCAM, neural cell adhesion molecule; NOVA-1, Neuro-oncological ventralantigen 1NSG, nod scid gamma (species of immunocompromised mouse); OAT,ornithine aminotransferase; ORGANOID, floating aggregate of cellscomprised of both epithelia and mesenchymal cells; PAK3,Serine/threonine-protein kinase PAK 3; PCSK1, proprotein convertase 1involved in processing of hormones; PDX1, pancreatic and duodenalhomeobox 1; PBGs, peribiliary glands, stem cell niches for biliary treestem cells; PDX, patient-derived xenograft; PHACTR2, phosphatase andactin regulator 2; RPS6KA2 Ribosomal protein S6 kinase alpha-2; SBHA,suberic bis-hydroxamic acid; SAHA, suberoylanilide hydroxamic acid(potent, reversible class I and II HDAC inhibitor); SALL4, Sal-likeprotein 4; SLC16A14: membrane channel for solute carrier family 16(monocarboxylic acid transporters), member 14; SOX, Sry-related HMG box;SPHEROID, floating aggregate of cells that are only one cell type; TCGA,The Cancer Genome Atlas; TEM, transmission electron microscopy; TMA,tissue microarrays; TMEM163, Transmembrane protein 163 involved in zinctransport and homeostasis; TNRC6C, Trinucleotide repeat-containing gene6C protein involved in gene regulation; TROP-2, tumor-associated calciumsignal transducer 2; VEGF, vascular endothelial cell growth factor.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof necessary fee.

FIG. 1 shows the analysis of human FL-HCCs compared to normal adultlivers using tissue microarrays (TMAs) and immunohistochemistry (IHC)assays. (a) Hematoxylin/eosin stained paraffin sections of TMAs. (b, c)Representative IHC assays on sections from the original blocks of normaladult liver versus hFL-HCC tumors. (d) Complete results of IHC assays.Additional details are given in FIG. 7. The scale bar is 50 μm (a), 25μm (b-c).

FIG. 2 shows the characterization and tumorigenicity of thetransplantable human FL-HCC tumor line, TU-2010. (a, b)Hematoxylin/eosin stained sections of the original solid tumor found inthe liver. (c, d) Subcutaneous tumor generated in NSG mice. (e)Intraperitoneal transplants. (f) Histology of the centers (1) versusperimeters (2) of the subcutaneous tumor at low and high magnifications.Histology and IHC assays on the original hFL-HCC cells from the ascitestumor of TU-2010 are given in FIG. 9a . The scale bar=25 μm. (g)Proportion of human tumor cells to host cells in subcutaneous tumors.Flow cytometric analyses of tumors dispersed into single cellsuspensions indicated that the cell suspensions were over 50% and up to70% host cells. Shown is one in which the cell suspension was comprisedof 53.5% murine mesenchymal cells and the remainder are human tumorcells. By negatively sorting for cells positive for H-2K^(d), theenrichment of the human tumor cells reached above 95% routinely. (h)Proportion of human tumor cells to host cells in intraperitoneal tumorsof TU-2010. With intraperitoneal transplantation the passaging can bedone every about 8 weeks. Flow cytometry contrasting side scatter (SCC)versus expression of H-2K^(d) in hFL-HCCs derived from the ascites cellsfrom the peritoneum of mice. The murine ascites fluid and the cellsbound to the serosal surfaces comprise about 3.5% tumor cells and over96% host cells indicating the extraordinary extent of desmoplasticresponse with intraperitoneal transplants. (i) Limiting dilutiontumorigenicity assays of hFL-HCCs from TU-2010. The original tumorsample includes 4 liters of ascites tumor cells that were centrifuged,plated onto culture plastic and in serum-free Kubota's Medium (KM) forseveral weeks. Phase images of the original cultures are given in FIGS.4a and 4b (see also FIG. 10). Culture selection for endodermalstem/progenitors was performed. The tumors from the initial passageappeared in about 5 months. Subsequently tumors appeared by about 3months if about 10⁶ to 10⁷ tumor cells were transplanted in KMsupplemented with hyaluronans, HGF and VEGF. At passage 8, the tumorcells were dispersed, and the host mesenchymal cells depleted by sortingnegatively for cells positive for H-2K^(d). The purified hFL-HCC cellswere transplanted subcutaneously at cell numbers from 10² to 10⁶. At allconcentrations from 10⁵ cells and higher tumors formed in 100% of themice by 3 months; at 10³-10⁴ cells, all formed tumors within 4-5 months.At 100 cells, one tumor formed at 5 months; one at 6 months; and one by9 months.

FIG. 3 shows the results of IHC and flow cytometric assays on thexenotransplantable tumor line, TU-2010. (a) Representative FACScharacterizations of sorted hFL-HCC cells (cell suspensions depleted ofmurine host cells) from TU-2010. LGR5⁺ cells accounted for 68.9% of thecells in the tumors. Other antigens that were expressed by a significantpercentage of the cells from TU-2010 included CD44, the hyaluronanreceptor (61.4%); CD49f (25.4%); Signal transducer CD24 (32.9%); CD13,alanine aminopeptidase (12.5%); c-KIT (12.0%); E-cadherin (12.0%); andoncostatin M receptor (OSMR) (10.7%). Other antigens found routinely ina smaller percentage of cells included CXCR4, also called fusin or CD184(4.8%), EpCAM (4.3%), CD133, also called prominin (2.3%), TROP-2 (1.4%);and ICAM intercellular adhesion molecule (0.5%). (b) The hFL-HCCs ofTU-2010 were depleted of murine cells and sorted for LGR5⁺ cells by flowcytometry. Of these, only 1.1% were also EpCAM⁺. (c) IHC assay on thesorted LGR5⁺ cells demonstrated strong expression of LGR5 and an absenceof EpCAM. (d, e, f, g) Paraffin sections (5 m) from hFL-HCC tumors(subcutaneous) were prepared and subjected to IHC assays for an array ofantigens. Of those shown, all were positive with the exceptions ofEpCAM, alpha-fetoprotein (AFP), and MUC6. The survey comprised assaysfor endodermal stem/progenitor transcription factors and markers: SOX17,SOX9, CD44, LGR5, CK7 and CK19 (d); pluripotency genes and genesindicative of self-replication: NANOG, OCT4, KLF4, SOX2, BMI1 and SALL4(e); hepatic and other markers: SHH, EpCAM (essentially negative), CD68,HepPar-1, CK18, AFP (negative) (f); and pancreatic/endocrine markers:PDX1, NGN3, and MUC6 (negative), which was strongly expressed (g). CD68is a marker identified previously as routinely found in hFL-HCC cells.Additional assays and controls are given in FIG. 9b . The scale bar is25 Lm for all Figure panels.

FIG. 4 shows analysis of monolayer cultures of hFL-HCC cells fromTU-2010. (a) Suspensions of hFL-HCC cells from TU-2010 were plated ontoculture plastic and in serum-free KM. The cells transiently attached andformed star-like cells (see also FIG. 11). (b) Subsequently, the cellsbecame loosely attached to the dish and retained attachment to eachother via E-cadherin linkages such that they formed floating cell chains(“catena”). (c) If plated with KM supplemented with 2-5% FBS during theseeding phase and then converted to serum-free KM, the cells were ableto remain attached to the dishes for longer and formed colonies thatwere irregular and with extensions in diverse directions; theytransitioned into aggregates/spheroids that eventually floated into themedium. (d) IHC of these colonies indicated that they were uniformlypositive for stem cell traits such as LGR5 or NANOG. (e) The aggregates,but not monolayer cells, expressed low levels of EpCAM andcytokeratin19. Depletion of the host (murine) cells enabled hFL-HCCcells to form colonies at single cell seeding densities and that grewinto colonies within 2 weeks. After 2 weeks, these cells morphologicallyresemble the cultures of the original ascites cells. The scale bar is100 μm (a-e). (f) Using assays comparing invasion through filters coatedwith a basement membrane matrix versus uncoated filters, 100% of thehFL-HCCs from TU-2010 were able to invade compared to less than about70% of Huh7 cells, a liver cancer cell line. (g) A screen of drugeffects on monolayer cultures indicated that the hedgehog inhibitor,GDC-0449, had effects at 4 μM and especially at 20 μM to suppresssurvival and proliferation of hFL-HCC cells. Histone deacetylase (HDAC)inhibitors were more potent: SBHA at 10 μM and 20 μM and especially SAHAat 2 μM and 10 μM, had strong inhibitory effects on hFL-HCC cellsurvival and growth. Data are expressed as the mean±SD(**p<0.01,*p<0.05).

FIG. 5 shows analysis of spheroid cultures of the hFL-HCC cells fromTU-2010. Plating the hFL-HCC tumor cells from TU-2010 in serum-freeconditions on low attachment plates, and especially after depletion ofthe murine cells, resulted in spheroid formation. (a) The spheroids thatformed from freshly isolated hFL-HCC cells from TU-2010 and that weredepleted of murine cells (1^(st) spheroids) were sustainable in culturefor months and were able to be passaged to form secondary spheroids(2^(nd) spheroids). The scale bar is 100 μm. (b, c) Transmissionelectron micrographs (TEM) of the spheroids from TU-2010 (see also FIGS.11 and 12) indicate cell aggregates with some cell adhesion mechanisms;large numbers of secretory vesicles with electron-dense granules;partially formed ducts; and a wealth of mitochondria with aberrantcristae. The scale bar is 1 μm. (d) The HDAC inhibitors and GDC-0449were far more effective at strongly suppressing hFL-HCC survival andgrowth of the spheroid cultures than when the cells were in monolayercultures. Data are expressed as mean±SD (**p<0.01). (e) KM resulted inmaintenance of stemness in the cells. The differentiation mediacomprised serum-free hormonally defined medium (HDM) tailored forlineage restriction of normal hBTSCs to hepatocytes (HDM-H),cholangiocytes (HDM-C) or pancreatic islets (HDM-P). These media wereable to partially differentiate the hFL-HCCs from TU-2010; fullmaturation was not achieved due to the matrix-degrading enzymes producedby the tumor cells, which induced rapid dissolution (within hours) ofevery matrix substratum tested. The morphology of the colonies in KMversus the several differentiation media are shown. The changes occurredwere transient, as the cells transitioned rapidly towards spheroidformation. (f) During the few days when morphological changes wereobserved, there was an increase in expression of some markers associatedwith maturation to an adult fate. The example shown is CFTR, a trait ofmaturing or mature cholangiocytes. The scale bar is 100 μm. (g) qRT-PCRassays showed that stem cell traits (NANOG, POU5F1, SOX2, PROM1) weresuppressed in all the HDM. Surprisingly, so were KRT18 and PDX1 and to alesser extent KRT7. CD44 was partially suppressed in HDM-H and HDM-C butstrongly suppressed by HDM-P; LGR5 was suppressed in HDM-H and HDM-C butnot in HDM-P; TACSTD1 (EpCAM), KRT19, and CFTR were modestly suppressedin HDM-H and HDM-P, but EpCAM and especially CFTR were actually elevatedin HDM-C, and KRT19 was not affected. Data are expressed as mean±SD(**p<0.01, *p<0.05).

FIG. 6 shows the results of global gene expression analysis by RNA-seq.(a) A correlation heat map of gene expression profiles from RNAsequencing of adult hepatocytes (hAHEPs), biliary tree stem cells(hBTSCs), hepatoblasts (hHBs), and hepatic stem cells (hHpSCs) each fromthree different donors, as well as fibrolamellar hepatocellularcarcinoma (hFL-HCC) from four tumors in different passaged lines of thetransplantable tumor line, TU-2010. The tumor cells from TU-2010 weredepleted of host (murine) cells before being analyzed by RNA-seq. Valuesbetween 0 and 1 shown in each box correspond to the median pair-wisePearson correlation coefficient. All genes with an average normalizedexpected count >50 across all samples were included in the analysis(n=14,394). (b, c) Results of hierarchical clustering analysis based onEuclidian distance of gene expression profiles across the differentcategories of cells using either the 10,000 most highly expressed genes(b) or the 248 genes significantly differentially expressed betweenhBTSCs and hHpSCs (c). For both (b) and (c), only genes with an averagenormalized expected count >50 in at least one cell category wereconsidered. (d) Histograms of representative genes with distinctexpression patterns in hFL-HCCs are shown. These genes include anteriorgradient homolog 2 (AGR2), found expressed in other hFL-HCCs;Kruppel-like factors (KLF4 and KLF5), critical regulators of stemness;WNT7B, a member of the WNT (“wingless-related integration site”) familyof genes; doublecortin-like-kinase-1 (DCLK1), a marker of intestinaltumor stem cells; cytokeratin 20 (KRT20), found in intestinal andpancreatic cancers; MET, which encodes for the HGF receptor; arylhydrocarbon receptors (AHR), which can trigger malignant transformationof stem cells upon binding to dioxins and related agonists; and histonedeacetylase isoform 9 (HDAC9), which regulates chromatin accessibility.(e) Sashimi plot of RNA-seq read coverage and splice/fusion junctions(shown as arcs) for the fusion gene, DNAJB1-PRKACA, found only in thecells of the hFL-HCC transplantable tumor line. Solid peaks depict readsper kilobase per million reads mapped (RPKM). The fusion junction joinspart of exon 2 of DNAJB1 with the start of exon 2 of PRKACA. The fourreplicate samples of hFL-HCC tumors had 89, 139, 91, and 59 reads,respectively, that spanned the fusion junction. The fusion gene is notpresent in normal hBTSCs, hHpSCs, hHBs, or hAHEPs.

FIG. 7 shows the results of IHC assays on paraffin sections of theoriginal blocks. The IHC assays on paraffin sections of hFL-HCCs from 9donors indicated that all are positive for sonic hedgehog (SHH) and, ofthose assayed, all were also positive for HepPar-1. The majority of thetumors (7/9) were positive for SOX9 and PDX1, and 4/9 for BMI1. Therewere two distinct patterns of expression comprised of 1) ones in whichmost were positive for a given antigen (e.g. HepPar1, SHH, and SOX9) butwith heterogeneous levels of expression or 2) a pattern in which apercentage of the cells were positive (at least 20%) and the remaindernegative (e.g. PDX1 and BMI1).

FIG. 8 shows the results of IHC assays on the TMA samples of 18 hFL-HCCsversus 19 normal livers. (a) Tissue microarrays (TMAs) of normal humanliver versus (b) a human fibrolamellar hepatocellular carcinomas(hFL-HCC). (c) Table with summary of the number of positive versusnegative assays on the TMA samples. *With SALL4 staining, some paraffinsections were lost due to the buffer conditions used for antigenretrieval. The scale bar=25 μm.

FIG. 9 shows the results of IHC assays of the original tumor (ascites)versus the xenotransplantable tumor, TU-2010. (a) Cytology and IHCassays on cytospun ascites tumor cells. Cytology revealed smallaggregates of tumor cells with large pleomorphic nuclei and some formingpartial ductular structures. The IHC assays on TU-2010 indicated strongpositivity for endodermal stemness markers (SOX17, SOX9, PDX1, SALL4,and BMI1), hepatic markers (HepPar-1, CK7 and CK19), and CD68. The scalebar=50 μm. (b) Additional IHC assays of the xenotransplantable tumor. Inaddition to the assays shown in FIG. 3, other markers found to bestrongly positive included E-cadherin, NCAM, two forms of multidrugresistance genes (MDR1 and ABCG2), syndecan1 (heparan sulfateproteoglycan-1 or HS-PG-1), and VCAM-1. Controls for the IHC assays arealso provided. The scale bar=25 μm.

FIG. 10 shows images of monolayer cultures of the original (a) hFL-HCCcells versus the (b) xenotransplanted tumors of TU-2010. These are ofunsorted cells and, therefore, a mixture of host (murine) mesenchymalcells and the tumor cells. The scale bar=100 μm.

FIG. 11 shows TEM images of hFL-HCC spheroids from TU-2010 plated andmaintained in serum-free KM. (a) Tumor cells displayed numerousmicrovilli at their apical pole and tight junctions (arrow) at cell tocell contact, meaning the tumor cells could still polarize. Cells wererich in rough endoplasmic reticulum (RER) and Golgi apparatus (G). (b)At their apical pole, tumor cells had numerous secretory vesiclescontaining electron-dense (white arrows) or not electron-dense(asterisks) granules. Note the presence of microvilli. (c, d, e) Tumorcells were connected with tunneling nanotubes (TNT: dark arrows in c);Filopodia-like protrusions, which proceed TNT formation, were presentand established physical contact with neighboring cells (arrows in d ande). The scale bar correlates with different lengths in the differentimages: The scale bar=200 nm (a), 1 μm (b-e).

FIG. 12 shows TEM images of hFL-HCC spheroids from TU-2010 plated andmaintained in serum-free KM. (a, b) Cells were especially rich inmitochondria (oncocytic condition) with numerous pleomorphic,irregularly shaped, non-lucent mitochondria with irregular cristaedisorganization. G=Golgi apparatus. (c, d) Nuclei presented dispersedchromatin and large nucleoli implicating production of secretoryproteins. The scale bar=200 nm (a-b), 1 μm (c-d).

FIG. 13 shows the results of hierarchical clustering analysis. Allsamples were clustered based on the expression profiles of genessignificantly differentially expressed between hBTSCs and hHpSCs(n=248). The hFL-HCCs from TU-2010 clustered closest to hBTSCs andfurthest from hHpSCs. The hHBs and hAHEPs clustered more closely withhHpSCs than hFL-HCCs from TU-2010 and hBTSCs. Euclidean distance wasused as the clustering metric.

FIG. 14 shows expression data for representative cell type marker genes.RNA-seq normalized expected count data shown across all cell types forgenes that have previously been reported as markers of stemcells/progenitors (SOX9, FOXL1, CD44, ITGA6 (CD49f), and ITGB1 (CD29),hepatocytes (DLK1, AFP, HNF4A, CPS1, and APOB), biliary tree (KRT7,ITGB4 and ONECUT2), and pancreas (PDX1 and PCSK1). Error bars representstandard error of the mean.

FIG. 15 shows expression data for genes in the hedgehog signalingpathway. Log₂ relative expression value shown across all cell types forgenes in the hedgehog signaling pathway. Error bars represent standarderror of the mean.

FIG. 16 shows expression data for genes encoding histone deacetylases.Log₂ relative expression value shown across all cell types for genesthat code for histone deacetylases. Histone deacetylase 9 (HDAC9) islost entirely in hFL-HCCs. Error bars represent standard error of themean.

FIG. 17 shows the results of pathway enrichment analysis. Results ofingenuity pathway analysis (IPA) are shown for genes significantlydifferentially expressed between hFL-HCCs from TU-2010 and hBTSCs,hFL-HCCs and hHpSCs, and hBTSCs and hHpSCs.

FIG. 18 shows images of normal peribiliary glands. The biliary tree isreplete with peribiliary glands found throughout the duct wall(intramural glands) and others attached by tethers to the bile ductsurface (extramural glands). Those within the duct wall contain cells ofvarying phenotypic traits that are found to be in a pattern indicating amaturational lineage. (a) Hematoxylin/eosin stained section of biliarytree and showing the peribiliary glands. (b) Radial axis of maturationlineage of biliary tree cells. The most primitive biliary tree stemcells (stage 1-hBTSCs) are located deep within the walls of the bileducts and near the fibromuscular layer. These cells do not express LGR5or EpCAM but do express pluripotency genes (e.g. OCT4, SOX2, KLF4,NANOG) and endodermal stem cell markers (e.g. SOX9, SOX17, PDX1). As onemoves towards the lumen of the bile duct, the cells lose the stem celltraits and gradually acquire mature cell traits. At intermediate stagesin this process, the cells acquire LGR5 (stage-2-hBTSCs) and then EpCAM(stage-3-hBTSCs). At the lumen, no stem cell traits are found butinstead only markers of mature cells. If near the liver, those markersare hepatic; if near the pancreas, the markers are pancreatic; ifin-between, the markers are those of bile ducts. (c) Sonic hedgehogexpression in stage-3-hBTSCs. The scale bar=100 μm.

FIG. 19 shows images of cultures of normal biliary tree stem cells.Cultures of stage 2 of stage-3-hBTSC stages are achievable by platingonto culture plastic (or hyaluronans) and in serum-free Kubota's Medium.These conditions have not yet proven successful for stage-1-hBTSCs.Stage-2-hBTSCs form colonies of cells that are undulating and highlymotile (a). They remain so in the cholangiocyte hormonally definedmedium (HDM-C) as shown in (b). They do not express EpCAM (or CK19) onthe cells within the centers of the colonies but have slightly largercells at the perimeters of the colonies and that do express these traits(albeit muted relative to that seen in the stage-3-hBTSC colonies). (c)stage-3-hBTSCs form carpet-like colonies in which every cell expressesEpCAM (and also CK19). (d) The stage 2 and 3 colonies and theintermediates between them all express sonic hedgehog. The scale bar=50μm.

FIG. 20 shows a chart of known biliary tree stem cell populations andtheir lineage connections and the probable normal counterpart to thehFL-HCC tumor cells from the transplantable tumor line, TU-2010.

FIG. 21 shows hierarchical clustering based on RNA expression of 163genes differentially expressed between FL-HCC tumor cells (FLC) and bothhepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) uniquelyclusters FLC samples.

FIG. 22 shows RNA expression within a 16 gene subset of the 163differentially expressed genes most uniquely distinguishes FLC from HCCand CCA as well as non-tumor liver (Liver) and cholangiocytes (Chol).

FIG. 23 shows validation of the most uniquely expressed 16 genes in FLCin an independent FLC cohort (Simon). Primary tissues shown aboveinclude The Cancer Genome Atlas (TCGA) FLC (FLC), validation FLC set(Simon FLC), TCGA HCC, TCGA CCA, TCGA non-tumor liver (Liver),validation set non-tumor liver (Simon Liver) and TCGA non-tumorcholangiocytes (Chol).

FIG. 24 depicts RNA expression within a 16 gene subset of the FLC genesignature distinguishes purified FLC tumor cells from biliary tree stemcells, the likely cell type of origin, and other cell types within theliver. Cell types shown are purified FLC tumor cells from apatient-derived xenograft (FLC PDX), biliary tree stem cells (BTSC),hepatic stem cells (HpSC), hepatoblasts (HB), and adult hepatocytes(AHEP).

FIG. 25 depicts RNA expression within a 16 gene subset of the FLC genesignature distinguishes FLC from 23 other tumor types.

FIG. 26 summarizes Gene Ontology Molecular Function Analysis results ofthe 165 hFL-HCC gene signature showing enrichment in kinase activity,growth factor binding, and cyclic adenosine monophosphate (cAMP)binding.

FIG. 27 depicts Kinase Enrichment Analysis results of the 165 hFL-HCCgene signature showing enrichment in substrate targets of Protein kinaseA catalytic subunit alpha (PRKACA)

FIG. 28 summarizes the results from Protein-Protein Interaction (PPI)Hub Protein analysis and shows PRKACA and sarcoma (SRC) gene mayfunction as network hubs in hFL-HCCs.

DETAILED DESCRIPTION

Fibrolamellar carcinomas (FLCs), also referred to as fibrolamellarhepatocellular carcinomas (FL-HCC), occur primarily in children andyoung adults without evidence of any chronic disease. FLCs wererecognized only recently, within the last approximately 45 years, butnow account for approximately 1-5% of liver cancers worldwide. Theepidemiological factors remain unknown. To date, FLCs are treatable onlyby surgery, which however is unproductive if there is metastatic diseaseat the time of diagnosis. Other forms of therapy, such as chemo- andexternal radiation therapies, or specific drugs commonly used forhepatocellular carcinomas (HCCs), have proven ineffective for FLCs. Theaverage time to death post-diagnosis for FLC patients is only about 18months. This disclosure is related to tools for studying the disease(e.g., a transplantable tumor line), including drug screening andtesting, as well as methods of suppressing the growth of fibrolamellarhepatocellular carcinomas (FL-HCC).

Embodiments according to the present disclosure will be described morefully hereinafter. Aspects of the disclosure may, however, be embodiedin different forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art. Theterminology used in the description herein is for the purpose ofdescribing particular embodiments only and is not intended to belimiting.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the present applicationand relevant art and should not be interpreted in an idealized or overlyformal sense unless expressly so defined herein. While not explicitlydefined by below, such terms should be interpreted according to theircommon meaning.

The terminology used in the description herein is for the purpose ofdescribing particular embodiments only and is not intended to belimiting of the invention. All publications, patent applications,patents and other references mentioned herein are incorporated byreference in their entirety.

The practice of the present technology will employ, unless otherwiseindicated, conventional techniques of tissue culture, immunology,molecular biology, microbiology, cell biology, and recombinant DNA,which are within the skill of the art. See, e.g., Sambrook and Russelleds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; theseries Ausubel et al. eds. (2007) Current Protocols in MolecularBiology; the series Methods in Enzymology (Academic Press, Inc., N.Y.);MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press atOxford University Press); MacPherson et al. (1995) PCR 2: A PracticalApproach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual;Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique,5th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No.4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization;Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds.(1984) Transcription and Translation; Immobilized Cells and Enzymes (IRLPress (1986)); Perbal (1984) A Practical Guide to Molecular Cloning;Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells(Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer andExpression in Mammalian Cells; Mayer and Walker eds. (1987)Immunochemical Methods in Cell and Molecular Biology (Academic Press,London); and Herzenberg et al. eds (1996) Weir's Handbook ofExperimental Immunology.

Unless the context indicates otherwise, it is specifically intended thatthe various features of the invention described herein can be used inany combination. Moreover, the disclosure also contemplates that in someembodiments, any feature or combination of features set forth herein canbe excluded or omitted. To illustrate, if the specification states thata complex comprises components A, B and C, it is specifically intendedthat any of A, B or C, or a combination thereof, can be omitted anddisclaimed singularly or in any combination.

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 1.0 or 0.1, as appropriate, oralternatively by a variation of +/−15%, or alternatively 10%, oralternatively 5%, or alternatively 2%. It is to be understood, althoughnot always explicitly stated, that all numerical designations arepreceded by the term “about”. It is to be understood that such rangeformat is used for convenience and brevity and should be understoodflexibly to include numerical values explicitly specified as limits of arange, but also to include all individual numerical values or sub-rangesencompassed within that range as if each numerical value and sub-rangeis explicitly specified. For example, a ratio in the range of about 1 toabout 200 should be understood to include the explicitly recited limitsof about 1 and about 200, but also to include individual ratios such asabout 2, about 3, and about 4, and sub-ranges such as about 10 to about50, about 20 to about 100, and so forth. It also is to be understood,although not always explicitly stated, that the reagents describedherein are merely exemplary and that equivalents of such are known inthe art.

DEFINITIONS

As used herein, the singular terms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to a cell can include multiple cells unless thecontext clearly dictates otherwise.

The term “about,” as used herein when referring to a measurable valuesuch as an amount or concentration (e.g., the percentage of collagen inthe total proteins in the biomatrix scaffold) and the like, is meant toencompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of thespecified amount.

The terms or “acceptable,” “effective,” or “sufficient” when used todescribe the selection of any components, ranges, dose forms, etc.disclosed herein intend that said component, range, dose form, etc. issuitable for the disclosed purpose.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

As used herein, the term “aggregates” refer to a plurality of cells thatare amassed together. The aggregates may vary in both size and shape ormay be substantially uniform in size and/or shape. The cell aggregatesused herein can be of various shapes, such as, for example, a sphere, acylinder (preferably with equal height and diameter), or rod-like amongothers. Although other shaped aggregates may be used, in one embodimentof the disclosure, it is generally preferable that the cell aggregatesbe spherical or cylindrical. If the aggregates are comprised of only onecell type, they are referred to as “spheroids; if they are a mixture ofmultiple cell types (e.g. epithelia and mesenchymal cells), they arereferred to as “organoids.” In addition, the term “spheroid” indicates afloating aggregate of cells all being the same cell type (e.g. anaggregate from a cell line); an “organoid” is a floating aggregate ofcells comprised of multiple cell types, an epithelial cell and itsmesenchymal partner cells, typically an endothelial cell and a stromalcell. The cells can be stem/progenitors of these categories of cells orcan be mature cells.

As used herein, the term “cell” refers to a eukaryotic cell. In someembodiments, this cell is of animal origin and can be a stem cell or asomatic cell. The term “population of cells” refers to a group of one ormore cells of the same or different cell type with the same or differentorigin. In some embodiments, this population of cells may be derivedfrom a cell line; in some embodiments, this population of cells may bederived from a sample of organ or tissue.

The term “biliary tree stem cells” refers to stem cells found throughoutthe biliary tree with the ability to transition into committed hepaticand/or pancreatic progenitor cells. They are found in both theextramural glands—tethered to the surface of the bile ducts—and theintramural glands—within the bile duct walls. The generic biomarkers forthe biliary tree stem cells (hBTSCs) include pluripotency genes (e.g.OCT4, SOX2, NANOG, SALL4, KLF4, KLF5); one or another of the isoforms(standard or variant) of CD44, the hyaluronan receptors; CXCR4;cytokeratins 8 and 18. There are 3 stages identified so far: stage 1hBTSCs: expresses sodium iodide symporter, CXCR4 but not LGR5 or EpCAM;stage 2 hBTSCs express less of NIS but gain expression of LGR5 but notEpCAM; stage 3 hBTSCs (found in the gallbladder, in the largeintrahepatic bile ducts and hepato-pancreatic common duct) expressesLGR5 and EpCAM and is a precursor to the hepatic stem cells (in theliver) and to the pancreatic stem cells (in the hepato-pancreatic commonduct.

As used herein the term “cancer stem cells” refers to the cells foundwithin solid tumors or hematological cancers that possesscharacteristics associated with normal stem cells, specifically theability to self-replicate and to be multipotent, that is give rise tomultiple cell types.

The term “hepatic stem cells” refers to stem cells found in the canalsof Hering, intrahepatic bile ductules, connecting the ends of thebiliary tree to the liver and retaining the ability to self-replicateand be multipotent. The biomarkers for these cells include epithelialcell adhesion molecule (EpCAM) found in the cytoplasm and at the plasmamembrane, neural cell adhesion molecule (NCAM), very low levels (if any)of albumin, an absence of alpha-fetoprotein (AFP), an absence of P450A7, an absence of secretin receptor (SR). Hepatic stem cells andhepatoblasts express cytokeratins 8 and 18 and 19.

The term “hepatoblasts” refers to bipotent hepatic cells that can giverise to hepatocytic and cholangiocytic lineages, that have anextraordinary ability to proliferate (that is expand) but with lessability to self-replicate than is observed in hepatic stem cells. Thesecells are characterized by a biomarker profile that overlaps with but isdistinct from hepatic stem cells, expressing EpCAM primarily at theplasma membrane, intercellular adhesion molecule (ICAM-1) but not NCAM,P450A7, cytokeratin 7, secretin receptor, albumin, high levels of AFP,and minimal (if any) pluripotency genes.

As used herein the term “committed progenitor” refers to a unipotentprogenitor cell that gives rise to a single cell type, e.g. a committedhepatocytic progenitor cell (usually recognized by expression ofalbumin, AFP, glycogen, ICAM-1, various enzymes involved with glycogensynthesis) gives rise to hepatocytes and a committed biliary (orcholangiocytic) progenitor (recognized by expression of EpCAM,cytokeratins 7 and 19, aquaporins, CFTR, and membrane pumps associatedwith management of bile) gives rise to cholangiocytes.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but do notexclude others. As used herein, the transitional phrase “consistingessentially of” (and grammatical variants) is to be interpreted asencompassing the recited materials or steps “and those that do notmaterially affect the basic and novel characteristic(s)” of the recitedembodiment. See, In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463(CCPA 1976) (emphasis in the original); see also MPEP §2111.03. Thus,the term “consisting essentially of” as used herein should not beinterpreted as equivalent to “comprising.” “Consisting of” shall meanexcluding more than trace elements of other ingredients and substantialmethod steps for administering the compositions disclosed herein.Aspects defined by each of these transition terms are within the scopeof the present disclosure.

The term “culture” or “cell culture” means the maintenance of cells inan artificial, in vitro environment. A “cell culture system” is usedherein to refer to culture conditions in which a population of cells maybe grown as monolayers or in suspension (spheroids, organoids).

“Culture medium” is used herein to refer to a nutrient solution for theculturing, growth, or proliferation of cells. Culture medium may becharacterized by functional properties such as, but not limited to, theability to maintain cells in a particular state (e.g. a pluripotentstate, a quiescent state, etc.), to mature cells—in some instances,specifically, to promote the differentiation of progenitor cells intocells of a particular lineage. Non-limiting examples of culture mediaare Kubota's medium, a medium designed for endodermal stem/progenitors,a hormonally defined medium (HDM) designed to drive the stem/progenitorseither to hepatocytes (HDM-H), to cholangiocytes (HDM-C), or topancreatic islets (HDM-P), which are further defined herein below. Insome embodiments the medium may be a “seeding medium” used to present orintroduce cells into a given environment. In other embodiments, themedium may be a “differentiation medium” used to facilitate thedifferentiation of cells. Such media are comprised of a “basal medium”or a mixture of nutrients, minerals, amino acids, sugars, lipids, andtrace elements and supplemented either with serum (serum supplementedmedia or SSM) or with a defined mix of purified hormones, growth factorsand nutrients (HDM) and used for maintenance of cells ex vivo. As usedherein, “HDM-H” is an HDM used in combination with substrata of type IVcollagen and laminin to drive the differentiation of endodermalstem/progenitors to mature hepatocytes. “HDM-C,” as used herein, refersto an HDM used in combination with substrata of type I/III collagen andfibronectin and designed to drive the differentiation of endodermalstem/progenitors to mature cholangiocytes. “HDM-P” is an HDM used incombination with substrata of type IV collagen and laminin to drive thedifferentiation of endodermal stem/progenitors to a mature pancreaticislet fate. Basal media are buffers comprised of amino acids, sugars,lipids, vitamins, minerals, salts, and various nutrients in compositionsthat mimic the chemical constituents of interstitial fluid around cells.Basal media are the starting points for buffers used for cell cultures.In addition, cell culture media are usually comprised of basal mediasupplemented with a small percentage (typically 2-10%) serum to providerequisite signaling molecules (hormones, growth factors) needed to drivea biological process (e.g. proliferation, differentiation). Although theserum can be autologous to the cell types used in cultures, it is mostcommonly serum from animals routinely slaughtered for agricultural orfood purposes such as serum from cows, sheep, goats, horses, etc. Serumis also used to inactivate enzymes that are part of tissue dissociationprocesses.

The terms “equivalent” or “biological equivalent” are usedinterchangeably when referring to a particular molecule, biological, orcellular material and intend those having minimal homology while stillmaintaining desired structure or functionality.

As used herein, the term “expression” refers to the process by whichpolynucleotides are transcribed into mRNA and/or the process by whichthe transcribed mRNA is subsequently being translated into peptides,polypeptides, or proteins. If the polynucleotide is derived from genomicDNA, expression may include splicing of the mRNA in a eukaryotic cell.The expression level of a gene may be determined by measuring the amountof mRNA or protein in a cell or tissue sample. Further, the expressionlevel of multiple genes can be determined to establish an expressionprofile for a particular sample. As used herein, the term “lower level”in reference to expression level refers to an amount in a cancer cellthat is less than the amount in a non-cancer control sample.

Exemplary growth factors include, but are not limited to, epidermalgrowth factors (EGFs), fibroblast growth factors (FGFs), hepatocytegrowth factors (HGFs), insulin-like growth factors (IGFs), transforminggrowth factors (TGFs), nerve growth factors (NGFs), neurotrophicfactors, interleukins, leukemia inhibitory factors (LIFs), vascularendothelial cell growth factors (VEGFs), platelet-derived growth factors(PDGFs), stem cell factor (SCFs), colony stimulating factors (CSFs),GM-CSFs, erythropoietin, thrombopoietin, heparin binding growth factors,IGF binding proteins, placental growth factors, Wnt signals.

As used herein, the term “functional” may be used to modify anymolecule, biological, or cellular material to intend that itaccomplishes a particular, specified effect.

The term “gene” as used herein is meant to broadly include any nucleicacid sequence transcribed into an RNA molecule, whether the RNA iscoding (e.g., mRNA) or non-coding (e.g., ncRNA).

As used herein, the term “hyaluronan,” or “hyaluronic acid,” refers to apolymer of a uronic acid and an aminosugar [1-3] composed of adisaccharide unit of glucosamine and gluronic acid linked by β1-4, β1-3bonds and salts thereof. Thus, the term hyaluronan refers to bothnatural and synthetic hyaluronan.

As used herein, the term “immunocompromised” in reference to an animalis one with an impaired immune system such that it is incapable of fullyreacting immunologically to pathogens. Those skilled in the art willrecognize that this may be due to a genetic disorder, disease process,irradiation or drugs, such as corticosteroids or immunosuppressiveagents, given to treat a disorder that inhibits immune function.Examples of drugs that suppress the immune system are methotrexate,cyclophosphamide, 6-mercaptopurine, vincristine, and the like. Suitableimmunocompromised animals for use in the practice of the presentdisclosure are the athymic nude mouse, SCID mouse, SCID/NOD, NOD scidgamma (NSG), BNX immunodeficient mouse, and the like, In one preferredembodiment, the host will be immunocompromised if the transplant ofhFL-HCC cells is allogeneic or xenogeneic

The term “isolated” as used herein refers to molecules or biologicals orcellular materials being substantially free from other materials.

“Kubota's medium” as used herein refers to any basal medium containingno copper, low calcium (<0.5 mM), insulin, transferrin/Fe, free fattyacids bound to purified albumin and, optionally, also high densitylipoprotein. Kubota's Medium or its equivalent is serum-free andcontains only purified and defined mix of hormones, growth factors, andnutrients. In certain embodiments, the medium is comprised of aserum-free basal medium (e.g., RPMI 1640 or DME/F12) containing nocopper, low calcium (<0.5 mM) and supplemented with insulin (5 μg/mL),transferrin/Fe (5 μg/mL), high density lipoprotein (10 μg/mL), selenium(10-10 M), zinc (10 12 M), nicotinamide (5 μg/mL), and a mixture ofpurified free fatty acids bound to a form of purified albumin.Non-limiting, exemplary methods for the preparation of this media havebeen published elsewhere, e.g., Kubota H, Reid L M, Proceedings of theNational Academy of Sciences (USA) 2000; 97:12132-12137, the disclosureof which is incorporated herein in its entirety by reference.

The term “mesenchymal cell” refer to cells of the mesenchyme. These areloose cells embedded in a mesh of proteins and fluid (i.e. extracellularmatrix). Mesenchyme gives rise to most of the body's connective tissues.

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides or analogsthereof. Polynucleotides can have any three dimensional structure andmay perform any function, known or unknown. The following arenon-limiting examples of polynucleotides: a gene or gene fragment (forexample, a probe, primer, EST or SAGE tag), exons, introns, messengerRNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes and primers.

A polynucleotide can comprise modified nucleotides, such as methylatednucleotides and nucleotide analogs. If present, modifications to thenucleotide structure can be imparted before or after assembly of thepolynucleotide. The sequence of nucleotides can be interrupted bynon-nucleotide components. A polynucleotide can be further modifiedafter polymerization, such as by conjugation with a labeling component.The term also refers to both double and single stranded molecules.Unless otherwise specified or required, any aspect of this technologythat is a polynucleotide encompasses both the double stranded form andeach of two complementary single stranded forms known or predicted tomake up the double stranded form.

The term “protein”, “peptide” and “polypeptide” are used interchangeablyand in their broadest sense to refer to a compound of two or moresubunit amino acids, amino acid analogs or peptidomimetics. The subunitsmay be linked by peptide bonds. In another aspect, the subunit may belinked by other bonds, e.g., ester, ether, etc. A protein or peptidemust contain at least two amino acids and no limitation is placed on themaximum number of amino acids which may comprise a protein's orpeptide's sequence. As used herein the term “amino acid” refers toeither natural and/or unnatural or synthetic amino acids, includingglycine and both the D and L optical isomers, amino acid analogs andpeptidomimetics.

As used herein, the term “subject” and “patient” are usedinterchangeably and are intended to mean any animal. In someembodiments, the subject may be a mammal. In further embodiments, thesubject may be a human or non-human animal (e.g. a mouse or rat).

As used herein, the terms “substantially,” “substantial,” and “about”are used to describe and account for small variations. When used inconjunction with an event or circumstance, the terms can refer toinstances in which the event or circumstance occurs precisely as well asinstances in which the event or circumstance occurs to a closeapproximation. For example, the terms can refer to less than or equal to±10%, such as less than or equal to ±5%, less than or equal to ±4%, lessthan or equal to ±3%, less than or equal to ±2%, less than or equal to±1%, less than or equal to ±0.5%, less than or equal to ±0.1%, or lessthan or equal to ±0.05%. In addition, when a cancer cell is said to“substantially not express” a particular gene, this refers to less thanor equal to ±10% (or more preferably less than or equal to ±5%) ascompared to a non-cancer cell.

The term “transplantable” in reference to a tumor line refers to a tumorgrown in a laboratory animal. The term “xenotransplantable” is one thathas or will be transplanted between members of different species, forexample, a human tumor that is transplantable into a mouse. A tumor froma donor animal (e.g. a human) is removed and often prepared into asingle-cell suspension and administered to the host/recipient animal(e.g. a mouse). Some tumors must be propagated by transplanting smallpieces of tumor or minced tumor material.

As used herein, “treating” or “treatment” of a disease in a subjectrefers to (1) preventing the symptoms or disease from occurring in asubject that is predisposed or does not yet display symptoms of thedisease; (2) inhibiting the disease or arresting its development; or (3)ameliorating or causing regression of the disease or the symptoms of thedisease. As understood in the art, “treatment” is an approach forobtaining beneficial or desired results, including clinical results. Forthe purposes of the present technology, beneficial or desired resultscan include one or more, but are not limited to, alleviation oramelioration of one or more symptoms, diminishment of extent of acondition (including a disease), stabilized (i.e., not worsening) stateof a condition (including disease), delay or slowing of condition(including disease), progression, amelioration or palliation of thecondition (including disease), states and remission (whether partial ortotal), whether detectable or undetectable.

Many embodiments described herein relate to a non-human animal that isimmunocompromised and able to be transplanted with human tumor cellswithout immunologically rejecting the human cells. Such a host is usedfor establishment of a transplantable human fibrolamellar hepatocellularcarcinoma (hFL-HCC) tumor line such as TU-2010, a non-human,immunocompromised animal carrying a transplantable human tumor offibrolamellar hepatocellular carcinoma cells; the tumor line is calledTU-2010.

The immunocompromised animal can be, for example, devoid of T cells,devoid of B cells, lacking functional NK cells, and/or deficient incytokine signaling. In some embodiments, the non-human animal is animmunocompromised mouse. The immunocompromised mouse can be, forexample, a NOD scid gamma mouse (or NOD. Cg-Prkdc^(scid)Il2rg^(tm1Wj1)/SzJ).

In some embodiments, the xenotransplanted tumor in the non-human animalis a subcutaneous tumor. In some embodiments, the xenotransplanted tumorin the non-human animal is an intraperitoneal tumor, an ascites. Thexenotransplanted tumor, once depleted of the host cells, can comprise,for example, approximately 10⁶ to 10⁷ hFL-HCC cells/gram

The hFL-HCC tumor line, TU-2010, is unusually rich in cancer stem cells.Whereas hepatocellular carcinomas (HCCs) are comprised typically of˜0.5-3% cancer stem cells, and cholangiocarcinomas (CCAs) are comprisedtypically of ˜10% cancer stem cells, the transplantable tumor line,TU-2010, is comprised of more than 60% cancer stem cells, a findingindicating the uniqueness of the TU-2010 tumor line.

The transplantable tumor line, TU-2010, is further comprised of a largepercentage of mesenchymal cells derived from the host, theimmunocompromised, non-human animal. The mesenchymal cells can comprise,for example, precursors to stellate cells, endothelial cells, stromalcells or pericytes. The non-human mesenchymal cells can account for 50%or more of the tumors transplanted subcutaneously and over 90% of thosetransplanted intraperitoneally.

The hFL-HCC cells of the TU-2010 transplantable tumor line express afusion transcript DNAJB1-PRKACA that has been found in the majority(˜70%) of human FL-HCC tumors.

The hFL-HCC cells of the TU-2010 transplantable tumor line do notexpress HDAC9.

The hFL-HCC cells of the TU-2010 transplantable tumor line express LGR5but substantially do not express or express negligible level ofepithelial cell adhesion molecule (EpCAM)

Human fibrolamellar hepatocellular carcinomas, including the cells inTU-2010, have phenotypic traits indicative of an origin from one oranother of the biliary tree stem cell (hBTSC) populations. These includeexpression of one or more of the following markers:

-   -   endodermal stem/progenitor transcription factors (SOX9, SOX17,        PDX1, FOXA1)    -   one or more stem cell genes such as the hyaluronan receptors        (CD44), SALL4, LGR5, TROP-2, prominin (CD133), and Sonic        Hedgehog (SHH)    -   one or more pluripotency genes and genes indicative of        self-replication selected from NANOG, OCT4, KLF4, KLF5, SOX2,        BMI1, AGR2 and SALL4.    -   one or more hepatic markers selected from CK7, CK18, CK19, CD68,        DCLK1, HepPar-1, and KRT20 (KRT20 is found also in the        epithelial cells of the intestine)    -   one or more pancreatic/endocrine markers such as PDX1, NGN3,        PCSK1.

Fibrolamellar hepatocellular carcinomas, including the FL-HCCs inTU-2010, express various markers indicative of malignancy. The TU-2010line expresses high levels of anterior gradient protein 2 homolog(AGR-2), associated with the down regulation of the phosphoprotein, P53,a tumor suppressor, and it secretes large amounts of enzymes thatdegrade extracellular matrix components. These findings are relevant toregulation of p53.

The hFL-HCC tumors, including the cells of the TU-2010 tumor line,express high levels of the aryl hydrocarbon receptor (AHR) implicating apossible pathogenic and oncogenic process involving dioxins ordioxin-like molecules.

The hFL-HCC tumors have aberrations or loss of expression of one (ormore) histone deacetylases. For example the TU-2010 tumor line's cellshave a lack of expression of HDAC 9, a member of a family of histonedeacetylases involved in epigenetic repression and involved inregulation of transcription, development and cell cycle. HDAC 9 servesdeacetylation of lysine residues on the N-terminal part of certain corehistones (H2A, H2B, H3 and H4).

The non-human host used for transplantable human tumors, such as thetransplantable human tumor line, TU-2010, is, of necessity, animmunocompromised host since the transplants are xenogeneic. Theimmunocompromised host can be, for example, devoid of T cells, devoid ofB cells, lacking functional NK cells, and/or deficient in cytokinesignaling. In some embodiments, the non-human host is animmunocompromised mouse. The immunocompromised mouse can be, forexample, a NOD scid gamma (NSG) mouse.

The tumor cells of the transplantable tumor line can be transplantedsubcutaneously or intraperitoneally in the non-human host.

The hFL-HCC tumors are rich in cancer stem cells. TU-2010 transplantabletumor line is particularly rich in human cancer stem cells. Whereashepatocellular carcinomas (HCCs) are comprised typically of ˜0.5-3%cancer stem cells, and cholangiocarcinomas (CCAs) are typically ˜10^(%)cancer stem cells, the TU-2010 transplantable tumor line is more than60% cancer stem cells.

The hFL-HCCs can be highly desmoplastic, meaning that the tumor cellselicit a strong reaction from mesenchymal cells located near to thetumor cells. The TU-2010 tumor line is representative of this ability ofhFL-HCCs to elicit desmoplastic responses; it comprises a mixture ofhFL-HCC cells and a large percentage of host (i.e. murine) mesenchymalcells that can comprise, for example, precursors to stellate cells andendothelial cells. The host mesenchymal cells can account for over halfof the cells of the tumor in subcutaneous tumors and over 90% of them inintraperitoneal tumors

The human FL-HCC cells of the tumor line, TU-2010, express the fusiontranscript DNAJB1-PRKACA, found in ˜80% of FL-HCC tumors.

The cells of the human transplantable tumor line, TU-2010, express LGR5but substantially do not express or express negligible levels of EpCAM.

In some embodiments, the human FL-HCC cells of the tumor line, TU-2010,express one or more markers indicative of an origin in one or another ofthe biliary tree stem cell subpopulations. These include one or more ofthe following biomarkers:

-   -   endodermal stem/progenitor transcription factors (SOX9, SOX17,        PDX1, FOXA1),    -   stem cell genes such as the hyaluronan receptors (CD44), LGR5,        TROP-2, prominin (CD133), and Sonic Hedgehog (SHH)    -   one or more pluripotency genes and genes indicative of        self-replication selected from NANOG, OCT4, KLF4, KLF5, SOX2,        BMI1, and SALL4    -   one or more hepatic markers selected from CK7, CK18, CK19, CD68,        DCLK1, and HepPar-1, and KRT20 (also found in the epithelial        cells of the intestine)    -   one or more pancreatic/endocrine markers such as PDX1, NGN3,        PCSK1.

The human FL-HCC cells, such as occurs in the TU-2010 tumor line,express biomarkers of malignancy. These include high levels of theanterior gradient protein 2 homolog (AGR-2), associated with the downregulation of the phosphoprotein, p53, a tumor suppressor and/or secretehigh levels of enzymes that degrade extracellular matrix components.

The human FL-HCC cells, such as those of the TU-2010 tumor line and inprimary FL-HCCs, express high levels of the aryl hydrocarbon receptor(AHR) implicating a possible pathogenic/oncogenic process involvingdioxins or dioxin-like molecules as contributing to the development ofthe TU-2010 tumor cells and other FL-HCCs.

Human FL-HCCs have aberrations in or loss of expression of one oranother of the histone deacetylase genes, a family of genes involved inregulation of transcription, development and the cell cycle throughenzymatic removal of acetyl groups from histones. For example, theTU-2010 tumor line's cells have a lack of expression of HDAC9, HDAC 9 isinvolved in deacetylation of lysine residues on the N-terminal part ofcertain core histones (e.g. H2A, H2B3, H3 and H4). HDAC9 is low inprimary FL-HCC tumors and also in HCCs as well.

Further embodiments relate to a tissue sample obtained from the tumorline.

hFL-HCC Cell Cultures.

Many embodiments described herein also relate to a cell culturecomprising human FL-HCC cells. Ideally the hFL-HCCs are maintained inculture in a serum-free medium. In some embodiments, the serum-freemedium is Kubota's Medium, one that culture selects for endodermalstem/progenitors and is non-permissive for cells at later maturationallineage stages.

Kubota's Medium is a wholly defined medium originally designed forrodent hepatoblasts and later found effective also human hepatoblasts(hHBs), human hepatic stem cells (hHpSCs), human biliary tree stem cells(hBTSCs), human pancreatic stem cells and for human hepatic andpancreatic progenitors. In some embodiments, Kubota's Medium comprisesany basal medium (e.g., RPMI 1640 or DMEM-F12) with no copper, lowcalcium (e.g., 0.3 mM), ˜10⁻⁹ M selenium, ˜0.1% bovine serum albumin orhuman serum albumin (highly purified and fatty acid free), ˜4.5 mMnicotinamide, ˜0.1 nM zinc sulfate heptahydrate, ˜10⁻⁸ M hydrocortisone,˜5 μg/ml transferrin/Fe, ˜5 μg/ml insulin, ˜10 μg/ml high densitylipoprotein, and a mixture of purified free fatty acids that are addedafter binding them to purified serum albumin. The free fatty acidmixture consists of ˜100 mM each of palmitic acid, palmitoleic acid,oleic acid, linoleic acid, linolenic acid, and stearic acid

In some embodiments used for maintaining the cells as stem cells, theserum-free medium is further supplemented with hyaluronans or substrataof hyaluronans are used.

In some embodiments when the cells are being differentiated towards anadult fate, the serum-free hormonally defined medium is comprised ofKubota's Medium is further supplemented with at least one growth factoror cytokine. The growth factor can be, for example, epidermal growthfactor (EGF), hepatocyte growth factor (HGF) fibroblast growth factor(FGF) and/or vascular endothelial cell growth factor (VEGF).

In some embodiments as when the cells are being differentiated towards ahepatocyte fate, the serum-free hormonally defined medium is comprisedof Kubota's Medium is further supplemented with: calcium to a level of˜0.6 mM; ˜10⁻¹²M copper; EGF (˜10 ng/ml); bFGF (˜20 ng/ml);tri-iodothyronine or T3 (˜10⁻⁹M); glucagon (7 μg/L), galactose (2 μg/L),oncostatin M (˜10 ng/ml); and HGF (˜20 ng/ml). For more optimaldifferentiation to an hepatocyte fate, this HDM is used in combinationwith embedding the cells into a mixture of type IV collagen, laminin,and hyaluronans.

In some embodiments as when the cells are being differentiated towards acholangiocyte fate, the serum-free hormonally defined medium iscomprised of Kubota's Medium that is further supplemented with: calciumto a level of ˜0.6 mM; ˜10⁻¹²M copper; bFGF (˜20 ng/ml); T3 (˜10⁻⁹M);VEGF (20 ng/ml) and HGF (10 ng/ml). For more optimal differentiation toa cholangiocyte fate, this HDM-C is used in combination with embeddingthe cells into a mix of type I collagen and hyaluronans.

In some embodiments as when the cells are being differentiated towards apancreatic islet fate, the serum-free hormonally defined medium iscomprised of Kubota's Medium is prepared without hydrocortisone and thenfurther supplemented with: calcium to a level of ˜0.6 mM; ˜10⁻¹² Mcopper; bFGF (˜20 ng/ml); B27 (˜2%), ascorbic acid (˜0.1 mM),cyclopamine (˜0.25 μM), retinoic acid (˜1 μM); furthermore, the bFGF isused for the first 4 days and then is replaced with exendin-4 (50 ng/ml)and HGF (20 ng/ml) for the remainder of the time. For more optimaldifferentiation to a pancreatic islet fate, this HDM-P is used incombination with embedding cells into a mix of type IV collagen,laminin, and hyaluronans

In some embodiments, the cultures are primary cultures of the dispersedtumor and so are a mix of the hFL-HCCs and the host (e.g. murine)mesenchymal cells.

In some embodiments, the human cells can be purified by immune-selectionfrom the tumor cell suspensions such that the cultures are predominantly(>90%) the human FL-HCC cells.

In some embodiments, the human FL-HCC cells of the cell cultures arerich in cancer stem cells. If the host (e.g. murine) mesenchymal cellshave been depleted by immune-selection technologies, the hFL-HCCs aredominant (>90% of the cells in culture) and comprised of cancer stemcells that are over 50% and up to 70% of the human cells in thecultures.

In some embodiments, the human FL-HCC cells are plated onto a culturesubstratum such as plastic; in some, the substratum can be a purifiedform of a matrix component (e.g. hyaluronan, a collagen, an adhesionmolecule such as laminin); in some, the substratum can be a complexextracellular matrix extract such as a biomatrix scaffold or Matrigel.

In some embodiments, the human FL-HCC cells of the cell culture can beestablished as spheroids or organoids, floating aggregates of cells. Inother embodiments, the hFL-HCC cells of the cell culture are in the formof monolayers.

Methods for Establishing and Maintaining hFL-HCC Tumor Lines

In some embodiments, the hFL-HCC cells are obtained from an ascitesfluid; in some embodiments, the hFL-HCC cells are obtained from a solidtumor from a patient suffering from the disease. To establish a tumorline, the tumor cells must first be culture selected for the cancerstem/progenitors present in the tumor. The culture selection makes useof restrictive conditions that are not permissive for other lineagestages of cells other than the ones desired. The method comprisesculturing cells obtained from the ascites fluid or from the solid tumorwith a wholly defined, serum-free media (e.g., Kubota's Medium),designed to culture select endodermal stem/progenitor cells (such as thecancer stem cells in the human FL-HCC tumors).

Many embodiments described herein also relate to a method for obtaininga human FL-HCC tumor line, comprising isolating human FL-HCC cells froma human subject suffering from FL-HCC; culture selecting the cancerstem/progenitor cell population(s) under conditions permissive for thecancer stem cells but not for the later maturational lineage stages ofcells; and then transplanting them into an immunocompromised, non-humananimal. The immunocompromised non-human animal can be, for example, animmunocompromised mouse such as NOD scid gamma (NSG) mouse.

In some embodiments, the hFL-HCC cells isolated from the human subjectare transplanted subcutaneously into the immunocompromised non-humananimal. In some embodiments, the hFL-HCC cells isolated from the humansubject are transplanted intraperitoneally into the immunocompromisednon-human animal.

In some embodiments, the method comprises transplanting from 10 to 10⁸hFL-HCC cells, or about 10² to 10⁷ hFL-HCC cells, or about 10² to 10³hFL-HCC cells, or about 10³ to 10⁴ hFL-HCC cells, or about 10⁴ to 10⁵hFL-HCC cells, or about 10⁵ to 10⁶ hFL-HCC cells, or about 10⁶ to 10⁷hFL-HCC cells, into the immunocompromised non-human animal. In someembodiments, the method comprises monitoring the immunocompromisednon-human animal for tumor formation for 2 to 9 months. Depending on thenumber of cells transplanted (with more transplanted cells correlatingwith more rapid tumor formation), for example, formation of axenografted human tumor can occur in about 2-3 months (withtransplantation of over 10⁶ cells), and ranging to 7-9 months (withtransplantation of 10-100 cells).

Many embodiments described herein also relate to a method formaintaining a human FL-HCC tumor line, comprising obtaining human FL-HCCcells from a first passaged xenografted tumor, dispersing the cells byenzymatic or mechanical methods into a cell suspension, andtransplanting the human FL-HCC cells into a second immunocompromisednon-human animal. The first and second immunocompromised non-humananimals can be, for example, immunocompromised mice such as NOD scidgamma mice.

In some embodiments, the method comprises culturing cells obtained fromthe xenografted tumor with a serum-free medium (e.g., Kubota's Medium)to culture select human FL-HCC cells. In some embodiments, theserum-free medium is supplemented with hyaluronans. In some embodiments,the serum-free medium is supplemented with one or more growth factors.The growth factors can be, for example, HGF, FGF, EGF, and/or VEGF.

In some embodiments, the human FL-HCC cells obtained from the firstimmunocompromised non-human animal are transplanted subcutaneously intothe second immunocompromised non-human animal. In some embodiments, thehuman FL-HCC cells obtained from the first immunocompromised non-humananimal are transplanted intraperitoneally into the secondimmunocompromised non-human animal.

Method for Culturing hFL-HCC Cells

Many embodiments described herein also relate to a method for culturinghuman FL-HCC cells, comprising separating human FL-HCC cells of axenografted tumor from non-human cells, suspending the separated humanFL-HCC cells in a serum-free medium (e.g., Kubota's Medium), and platingthe hFL-HCC cells onto culture plastic, or onto a purified matrixcomponent (e.g. hyaluronan, a collagen, an adhesion molecule such aslaminin) or into/onto a complex extracellular matrix extract (e.g.biomatrix scaffolds, Matrigel).

In some embodiments, the human FL-HCC cells of the xenografted tumor areseparated from non-human cells (e.g. murine mesenchymal cells) bymagnetic immuno-selection or by an equivalent immune-selectiontechnology (e.g. flow cytometry) or any technology that distinguisheshuman from non-human cells.

In some embodiments, the culture substratum comprises a culture plastic.In some embodiments, the culture substratum comprises a monolayercoating or a 3-dimensional form (e.g. hydrogel) of a purifiedextracellular matrix component (e.g. hyaluronan, a collagen, an adhesionmolecule such as laminin), a mix of one or more of the matrix component,or an extract enriched in extracellular matrix (e.g. biomatrixscaffolds, Matrigel).

In some embodiments, the human FL-HCC cells are suspended in the mediumand aggregation of the cells occurs to form spheroids if only thehFL-HCC are part of the aggregates or organoids if the aggregatescontain hFL-HCC and other cell types (e.g. vascular or mesenchymalcells). In some embodiments, the plated hFL-HCC cells form monolayers.

Method for Drug Screening and Testing

Many embodiments described herein also relate to a method for drugscreening, comprising contacting a candidate drug with cultured hFL-HCCcells, and monitoring the effect(s) of the candidate drug on thecultured hFL-HCC cells.

In some embodiments, the cultured hFL-HCC cells are in the form offloating aggregates of cells, spheroids or organoids.

Many embodiments described herein also relate to a method for drugtesting, comprising administering a candidate drug to a non-human animalthat has been transplanted with a tumor containing human FL-HCC cells,and monitoring the effect of the candidate drug on the xenotransplantedtumor.

Many embodiments described herein also relate to a method forsuppressing the growth of human FL-HCC cells, comprising treating thehFL-HCC cells with a hedgehog signaling pathway inhibitor and/or ahistone deacetylase inhibitor and/or some other candidate drug.

In some embodiments, the method comprises treating the human FL-HCCcells with a hedgehog signaling pathway inhibitor (e.g., GDC-0449). Insome embodiments, the method comprises treating the human FL-HCC cellswith a histone deacetylase inhibitor (e.g., SAHA or SBHA).

Many embodiments described herein also relate to a method for treatingFL-HCC in a human patient, comprising administering to the patient aneffective amount of a hedgehog signaling pathway inhibitor and/or ahistone deacetylase inhibitor.

In some embodiments, the method comprises administering to the patientan effective amount of a hedgehog signaling pathway inhibitor (e.g.,GDC-0449). In some embodiments, the method comprises administering tothe patient an effective amount of a histone deacetylase inhibitor(e.g., SAHA or SBHA).

Method for Diagnosis of hFL-HCCs

At present, the only biomarker identified for ˜80% of human FL-HCCs isthe fusion gene, DNAJB1-PRKACA. Histologically, the tumor isrecognizable as aggregates of large polygonal cells with abundanteosinophilic cytoplasm, large, vesiculated nuclei and large nucleoli;the tumor cells are nestled within bands of stroma. In addition to thefusion gene and the distinctive histological traits, one can identifyhFL-HCCs by phenotypic traits typical of biliary tree stem cells. ThesehFL-HCC tumors are hypothesized to derive from one or another of thebiliary tree stem cell subpopulations that constitute the native stemcells and progenitors for both liver and pancreas.

-   -   Recognition of the origins of hFL-HCCs from hBTSC subpopulations        is indicated by the expression of:        -   one or more endodermal transcription factors (e.g. SOX9,            SOX17, PDX1, FOXA1)        -   one or more pluripotency genes and genes indicative of            self-replication (e.g. NANOG, OCT4, KLF4, KLF5, SOX2, BMI1,            TROP-2, and SALL4) and evidence for multipotency        -   one or more hepatic markers (e.g. alpha-fetoprotein,            albumin, CK7, CK18, CK19, CD68, DCLK1, and HepPar-1), and            KRT20 (also found in the epithelial cells of the intestine)        -   one or more pancreatic/endocrine markers (e.g. PDX1, NGN3,            PCSK1, insulin, glucagon, amylase, MUC or mucin).    -   Recognition of malignancy in hFL-HCCs is indicated by:        -   Expression of high levels of anterior gradient protein 2            homolog (AGR-2), associated with the down regulation of the            phosphoprotein, p53, a tumor suppressor.        -   Release of high levels of enzymes that degrade and dissolve            extracellular matrix (e.g. heparanase, matrix            metalloproteinases-MMPs)        -   Aberrant regulation of p53, a tumor suppressor    -   Recognition of factors contributing to the pathogenic/oncogenic        process in hFL-HCCs is indicated by: high levels of the aryl        hydrocarbon receptor (AHR) implicating a possible contribution        of dioxins or dioxin-like molecules in the aetiology of hFL-HCCs    -   Recognition of key genes that are affected in hFL-HCCs is        indicated by: aberrations in or absence of one or more histone        deacetylases (HDAC), enzymes critically associated with        regulation of transcription, development, cell proliferation,        and the cell cycle.

In addition, the human FL-HCC tumors will have biomarkers of malignancy.Some of the biomarkers indicative of malignancy include high levels ofexpression of AGR2, over-production of matrix-degrading enzymes (e.g.heparanase, matrix metalloproteinases or MMPs), very high levels of AHRand aberrant levels of one or another HDAC gene and/or aberrations inthe regulation of p53, a tumor suppressor.

In some embodiment, the method further comprises culturing cellsobtained from an ascites fluid or solid tumor derived from a humansubject and subjecting it to culture conditions that are whollyserum-free and in a medium (e.g., Kubota's Medium), designed for cultureselection of endodermal cancer stem/progenitors and then detecting theformation of spheroids or organoids.

Methods for Determining Whether a Patient has FL-HCC and Treating aPatient Diagnosed with FL-HCC

In one aspect is provided a method of determining whether a patient hasfibrolamellar hepatocellular carcinoma (FL-HCC), comprising: (a)measuring gene expression levels of at least one of C10orf128, CA12,CREB3L1, GALNTL6, IRF4, ITPRIP, KCNE4, NOVA1, OAT, PAK3, PCSK1, PHACTR2,RPS6KA2, SLC16A14, TMEM163, and TNRC6C; and (b) comparing the geneexpression level to one or more control samples.

In some embodiments, overexpression of C10orf128, CA12, CREB3L1,GALNTL6, IRF4, ITPRIP, KCNE4, NOVA1, OAT, PAK3, PCSK1, PHACTR2, RPS6KA2,SLC16A14, TMEM163 or TNRC6C relative to the control sample is associatedwith presence of FL-HCC. On the other hand, a lack of increasedexpression of C10orf128, CA12, CREB3L1, GALNTL6, IRF4, ITPRIP, KCNE4,NOVA1, OAT, PAK3, PCSK1, PHACTR2, RPS6KA2, SLC16A14, TMEM163 or TNRC6Crelative to the control sample indicates that the patient is not likelyto have FL-HCC.

In other embodiments, overexpression of PCSK1, CA12, NOVA1, SLC16A14,TNRC6C, TMEM163, and RPS6KA2 relative to the control sample isassociated with presence of FL-HCC. On the other hand, a lack ofincreased expression of PCSK1, CA12, NOVA1, SLC16A14, TNRC6C, TMEM163,and RPS6KA2 relative to the control sample indicates that the patient isnot likely to have FL-HCC.

In yet other embodiments, overexpression of C10orf128, OAT, PAK3, PCSK1,PHACTR2, SLC16A14, TMEM163, and TNRC6C relative to the control sample isassociated with presence of FL-HCC. On the other hand, a lack ofincreased expression of PCSK1, CA12, NOVA1, SLC16A14, TNRC6C, TMEM163,and RPS6KA2 relative to the control sample indicates that the patient isnot likely to have FL-HCC.

In some embodiments, the control sample is selected from the groupconsisting of hepatocellular carcinomas (HCCs), cholangiocarcinomas(CCAs), normal liver cells, and normal cholangiocytes.

As used herein, the term “overexpression” refers to the level of mRNAand/or protein of a specific, for example, C10orf128, CA12, CREB3L1,GALNTL6, IRF4, ITPRIP, KCNE4, NOVA1, OAT, PAK3, PCSK1, PHACTR2, RPS6KA2,SLC16A14, TMEM163 or TNRC6C, expressed in a suspected tumor cell of asample collected from a patient being elevated in comparison to thelevel as measured in a control sample. The mRNA and/or proteinexpression level may be determined by a number of techniques known inthe art including, but not limited to, quantitative RT-PCR, westernblotting, immunohistochemistry, and suitable derivatives of the above.

In some embodiments, the gene expression of PCSK1 and at least oneadditional gene selected from the group consisting of C10orf128, CA12,CREB3L1, GALNTL6, IRF4, ITPRIP, KCNE4, NOVA1, OAT, PAK3, PHACTR2,RPS6KA2, SLC16A14, TMEM163, and TNRC6C is measured. In anotherembodiment, the gene expression of PCSK1 and at least one additionalgene selected from the group consisting of C10orf128, CA12, CREB3L1,GALNTL6, IRF4, ITPRIP, KCNE4, NOVA1, OAT, PAK3, PHACTR2, RPS6KA2,SLC16A14, TMEM163, and TNRC6C is overexpressed.

In some embodiments, at least two, at least three, at least four, atleast five, at least six, at least seven or all eight of genesC10orf128, CA12, CREB3L1, GALNTL6, IRF4, ITPRIP, KCNE4, NOVA1, OAT,PAK3, PCSK1, PHACTR2, RPS6KA2, SLC16A14, TMEM163 and TNRC6C aremeasured. In some embodiments, at least two, at least three, at leastfour, at least five, at least six, at least seven or all eight of genesC10orf128, CA12, CREB3L1, GALNTL6, IRF4, ITPRIP, KCNE4, NOVA1, OAT,PAK3, PCSK1, PHACTR2, RPS6KA2, SLC16A14, TMEM163 and TNRC6C areoverexpressed. In some embodiments, any one of the listed genes isexpressly excluded from the genetic signature of hFL-HCC.

In one aspect is provided a method of treating a patient determined tohave FL-HCC by administering to the patient an effective amount of atleast one therapeutic that decreases expression of at least one ofC10orf128, CA12, CREB3L1, GALNTL6, IRF4, ITPRIP, KCNE4, NOVA1, OAT,PAK3, PCSK1, PHACTR2, RPS6KA2, SLC16A14, TMEM163, or TNRC6C.

In some embodiments, the at least one therapeutic is selected from thegroup consisting of a small molecule, RNA interference, and a lockednucleic acid (LNA). In other embodiments, the at least one therapeuticis an immunotherapy.

In one aspect is provided a method of treating a patient determined tohave hFL-HCC by administering to the patient an effective amount of atleast one therapeutic that regulates PRKACA or SRC network hubs.

In another aspect is provided method of treating a patient determined tohave hFL-HCC by administering to the patient an effective amount of atleast one therapeutic that regulates substrate targets of the kinasePRKACA (Protein kinase A catalytic subunit alpha) or carbonicanhydrases.

Working Examples

Human fibrolamellar hepatocellular carcinomas (hFL-HCCs) are rare livercancers occurring in young people, have an unknown aetiology, and arecurrently treatable only by surgery. Immunohistochemistry (IHC) of 9hFL-HCCs and tissue microarrays of 18 FL-HCCs indicated robustexpression of endodermal stem/progenitor markers (SOX9, PDX1, SOX17,sonic hedgehog—SHH, SALL4, OCT4).

A first-ever, transplantable hFL-HCC tumor line, TU-2010, established inimmunocompromised mice, proved rich in cancer stem cells, indicatedfunctionally by spheroid/organoid formation, limiting dilutiontumorigenicity, and culture; by flow cytometric and IHC evidence ofpluripotency (e.g. KLF4, OCT4, NANOG) and endodermal stem/progenitormarkers (e.g., LGR5, SOX9, PDX1, CD44, SHH); and effects ofdifferentiation media on these traits. Transcriptomic analyses revealeda global expression profile for human FL-HCCs indicating theirderivation from biliary tree stem cells, stem cell precursors to liverand pancreas. A recurrent fusion gene unique to hFL-HCCs, DNAJB1-PRKACA,was confirmed. In vitro studies and phenotypic traits suggest hedgehogproteins and histone deacetylases to be therapeutic targets. Oikawa etal. (2015) Nature Communications 6:8070, incorporated by reference inits entirety.

Materials and Methods

1.1. Paraffin Sections of Normal Livers Versus hFL-HCCs.

Sections (5 μm) prepared from the original paraffin blocks of 9 hFL-HCCtumors and ones from Tissue MicroArrays (TMAs) of 19 normal adult liversand 18 hFL-HCC patients were obtained from Memorial Sloan KetteringCancer Center (MSKCC, New York City, N.Y.) and used for IHC assays. Theywere obtained with approval of the IRB at MSKCC. Handling of all thesamples fully met compliance and privacy requirements as per HIPAA laws.

1.2. Original Fibrolamellar Hepatocellular Carcinoma (hFL-HCC) Used toEstablish the Tumor Line, TU-2010.

A male patient, age 25, presented in August 2008 to Greenwich Hospital(Yale/New Haven Hospital, Greenwich, Conn.) with acute swelling of hisright lower leg. During the initial evaluations, he was found to have anextensive venous thrombus extending from his right ankle into hisinferior vena cava. A CT of his chest/abdomen/pelvis revealed multiplesmall pulmonary emboli and a large mass in his left liver with evidenceof metastatic disease in the perihepatic lymph nodes, omentum andperitoneum. He had a venous filter inserted above the thrombus, wasstarted on anticoagulation and transferred to MSKCC for further studiesand therapy. The patient underwent a liver biopsy resulting in apathologic diagnosis of hepatocellular carcinoma (HCC) and subsequentlyhad an extensive debulking procedure which included a left hepaticlobectomy and debulking of periotoneal and omental nodules.Macroscopically, the peritoneal and omentum nodules proved to be tumorsand histologically revealed tumor tissue consistent with a diagnosis ofhFL-HCC. Surgical pathology revealed tumor cells positive for HepPar1and cytokeratin 7 (CK7). Analyses of EMA (epithelial membrane antigen)and AFP (t-fetoprotein) were not conclusive and neither were tissuesstained for reticulin, iron, or PAS-3.

A summary of the characterization of the original tumor by thepathologists at Memorial Sloan Kettering Cancer Center (MSKCC) is givenin Table 1. A later round of biopsies resulted in similar pathologyreports. These included cytology on pleural fluid found replete withtumor cells, which also had a pathology consistent with a diagnosis ofFL-HCC.

TABLE 1 Summary of findings on the original hFL-HCC tumor used toestablish the TU-2010 transplantable tumor line. (patient data fromMemorial Sloan Kettering Cancer Center-MSKCC)** Staining Intensity (%cells Staining) or Gene Expression BIOMARKERS ASSAYS Change CommentsRibonucleotide IHC 2 + (80%) Certain drugs, such as reductase subunit M1gemcitabine, are of little benefit (RRM1) due to high expression of RRM1Breast Cancer IHC 2 + (75%) Obviates usefulness of cisplatin ResistantProtein and carboplatin (BCRP) Secreted Protein Acidic IHC 2 + (35%)Nab-paclitaxel and Rich in Cysteine (SPARC) SPARC Microarray Increased(3.51) Nab-paclitaxel Multidrug Resistance IHC 2 + (30%) Minimal effectsexpected with associated Protein 1 etoposide, vincristine (MRP1) ABCC1Microarray Increased (3.03) Minimal benefit with Paclitaxel, TopotecanHer2/Neu IHC 2 + (10%) and 1 + FISH analyses were done, and 60 (30%)interphase nuclei were examined; the ratio of HER2/neu signals tochromosome 17 signals was 1.63 to 1 indicating no amplification of thisgene HIF1A Microarray Increased (10.66) Agents associated with clinicalbenefits include sorafenib, sunitinib, bevacizumab PDGFRB MicroarrayIncreased (2.3) Agents associated with clinical benefits includesorafenib, sunitinib, imatinib TOP2B Microarray Increased (4.55)Beneficial agents include doxorubicin, epirubicin, liposomal doxorubicinADA Microarray Increased (4.05) Beneficial agents include pentostatinEstrogen receptor, Negative Classic hormone therapies are notprogesterone receptor, logical for use with the tumor androgen receptor**Approval for the research studies on the tumor and on the patient wasgiven by the IRB at MSKCC (New York City, NY), and compliance with HIPAAregulations was met.

The patient was subsequently treated with various oncoloytic agentsincluding sorafenib, doxorubicin, gemcitabine, cisplatinum, 5-FU,bevacizumab, and thalidomide with limited or no response. In Septemberof 2009 after showing progressive enlargement in the perihepatic andretroperitoneal nodes, recurrent disease in the liver, and increasingsize of omental and peritoneal nodules, the patient returned to MSKCC toobtain further tissue for tumor sensitivity studies and debulking.Biopsies were taken, but his disease was too extensive for debulking.Paclitaxel and thalidomide were then started based on sensitivitystudies but was poorly tolerated with continued disease progression, sotreatment was stopped. After 4 months it was realized that he had widelydisseminated disease especially in the ascites fluid. In early February2010, a palliative paracentesis was done for massive ascites, andapproximately 5 liters of fluid were removed and transferred to severalresearchers, including those in the UNC research lab, in hopes thatstudies on the tumor might identify alternate treatments. A week laterthe patient passed away peacefully.

1.3. Ascites Fluid.

Four liters of ascites fluid were received at UNC within 10 hours ofremoval from the patient. The cells were centrifuged and pooled,yielding about 2×10⁷ cells, and plated on plastic or other substrata(laminin, hyaluronans, types I, III or IV collagens) in serum-freeKubota's Medium (KM) prepared in either RPMI 1640 or in DMEM-F12 andpresented as two-dimensional (2D) monolayers or three-dimensional (3D)hydrogels. Serum-free Kubota's Medium (KM) has been found to select forendodermal stem cells and progenitors and is not permissive for survivalof mature cells. Culture selection for tumor cells with stem cellproperties was done in monolayer (2D) cultures and did best on plasticand in KM in DMEM-F12. Those in 3-D hydrogels behaved similarly in KMprepared in either DMEM-F12 or RPMI 1640, grew more slowly, and, inparallel, caused dissolution of hydrogels by hFL-HCC's considerableenzyme secretions that degrade extracellular matrix. This cultureselection process proved successful for establishment of thetransplantable tumor line as clarified in further details below.

1.4. Culture Conditions.

All media were sterile-filtered (0.22-μm filter) and kept in the dark at4° C. before use. Hyaluronans were obtained from Glycosan Biosciences(Salt Lake City, Utah; now part of Biotime, Alameda, Calif.). Type IIIand IV collagens and laminin were obtained from Becton Dickinson(Research Triangle Park, N.C.).

1.5. Kubota's Medium.

(KM) is a serum-free medium designed originally for rodent hepatoblastsand then found effective also for human hepatoblasts, hepatic stem cells(hHpSCs), biliary tree stem cells (hBTSCs), and for pancreaticprogenitors. It contains any basal medium (here being RPMI 1640) with nocopper, low calcium (0.3 mM), 10⁻⁹ M selenium, 0.1% BSA, 4.5 mMnicotinamide, 0.1 nM zinc sulfate heptahydrate, 10⁻⁸ M hydrocortisone, 5μg/ml transferrin/Fe, 5 μg/ml insulin, 10 μg/ml high densitylipoprotein, and a mixture of purified free fatty acids that are addedafter binding to purified human serum albumin. Kubota's Medium isavailable commercially from PhoenixSongs Biologicals (Branford, Conn.).

1.6. Hormonally Defined Media (HDM).

Supplements can be added to KM to generate a serum-free, hormonallydefined medium (HDM) that will facilitate differentiation of the normalhepatic or biliary tree stem cells to specific adult fates. Theseinclude supplementation with calcium to achieve at or above 0.6 mMconcentration, 1 nM tri-iodothyronine (T3), 10⁻¹² M copper, 10 nM ofhydrocortisone and 20 ng/ml of basic fibroblast growth factor (bFGF).The medium conditions over and above these needed to selectively yieldhepatocytes (HDM-H) versus cholangiocytes (HDM-C) versus pancreaticislets (HDM-P) are:

(1) HDM-H: supplementation further with 7 μg/L glucagon, 2 μg/Lgalactose, 10 ng/ml epidermal growth factor (EGF) and 20 ng/mlhepatocyte growth factor (HGF).(2) HDM-C: supplementation further with 20 ng/ml vascular endothelialcell growth factor (VEGF) 165 and 10 ng/ml HGF.(3) HDM-P: prepared without glucocorticoids and further supplementedwith 1% B27, 0.1 mM ascorbic acid, 0.25 μM cyclopamine, 1 μM retinoicacid, 20 ng/ml of FGF-7 for 4 days, then changed with one supplementedwith 50 ng/ml exendin-4 and 20 ng/ml of HGF for 6 more days ofinduction.

1.7. Tissue Sourcing of Normal Tissue.

Adult, normal, human biliary tissues were dissected from intact liversand pancreases obtained but not used for transplantation into a patient.They were obtained through organ donation programs via United Networkfor Organ Sharing (UNOS). Those used for these studies were considerednormal with no evidence of disease processes. Informed consent wasobtained from next of kin for use of the tissues for research purposes,protocols received Institutional Review Board approval, and processingwas compliant with Good Manufacturing Practice. The research protocolwas reviewed and approved by the Institutional Review Board for HumanResearch Studies at the University of North Carolina at Chapel Hill,N.C., USA.

1.8. Animals.

In preliminary studies, immunocompromised mice of several species (e.g.athymic nudes, SCID/NODs, NSGs) were obtained from suppliers or wereobtained from breeding colonies on the UNC campus and used as hosts forthe human FL-HCC cells. The findings were most successful withNOD.Cg-Prkdc^(scid) Il2rg^(tm1Wj1)/SzJ. These are known commonly as NODscid gamma or NSGs. These mice are devoid of T or B cells, lackfunctional NK cells and are deficient in cytokine signaling. The straincombines the features of the NOD/ShiLtJ (Stock Number 001976)background, the severe combined immune deficiency mutation (scid, whichis caused by a spontaneous mutation in the Prkdc gene), and the IL2receptor gamma chain deficiency. The animals were maintained in thequarters maintained by the Division of Laboratory Animals (DLAM).Procedures were performed according to protocols approved by the UNCSchool of Medicine at Chapel Hill IACUC. All species were inbred andhoused in UNC's DLAM sterile facility in micro-isolated autoclaved cageswith free access to autoclaved water and radiation sterilized food.

1.9. Transplantation of the Human FL-HCC Cells.

Efforts to establish a tumor line by transplanting the original ascitestumor cells immediately after removal from the patient were notsuccessful. Rather, success was achieved with cells that survived and sowere culture selected in serum-free Kubota's Medium (KM) and on cultureplastic or on a substratum of hyaluronans. The culture-selected tumorcells were transplanted and yielded tumors after an initial passage ofmore than 6 months in the NSG mice. Thereafter, xenografted establishedtumors passaged by mincing tumor in KM supplemented with 1% hyaluronans(uncross-linked) and further supplemented with 50 ng/ml each of HGF andVEGF. The tumor mince (approximately 20 mgs) in the KM+1%hyaluronans+growth factors was injected subcutaneously into mice. Thetumor mince form tumors in the absence of hyaluronans and growth factorsbut do so more slowly and will not yield tumors at all in some mice.Consistent, reproducible tumor formation occurred with the use of thesupplements. If transplanted intraperitoneally, the tumor cells spreadonto the serosal surfaces throughout the peritoneum and also onto theliver and pancreas.

Tissue processing of the human FL-HCC tumors to generate cellsuspensions for ex vivo studies was conducted in RPMI 1640 supplementedwith 0.1% bovine serum albumin, 1 nM selenium and antibiotics. Enzymaticprocessing buffer contained 600 U/ml type IV collagenase and 0.3 mg/mldeoxyribonuclease at 32° C. with frequent agitation for 15-20 min.Enriched suspensions were pressed through a 75 gauge mesh and spun at1200 RPM for 5 min before re-suspension. Estimated cell viability bytrypan blue exclusion was routinely higher than 95%.

1.10. Magnetic Immunoselection of Cells.

Human tumor cells were isolated from xenografted tumors. Negativesorting was done using EasySep magnetic bead immunoselection using themagnetic cups and beads (StemCell Technologies, Vancouver, Canada) andaccording to the manufacturer instructions. Briefly the dissociatedcells were washed in phosphate-buffered saline (PBS) with 3% fetalbovine serum (FBS) (staining medium) were treated with FcR blockingantibody and incubated with a cocktail of biotin-conjugated anti-mouseantibody against lineage cells (Miltenyi Biotec Inc, Auburn, Calif.),and with biotin-conjugated anti-mouse-MHC class I (H-2K^(d)) (clone;SF1-1.1) and -CD31 (clone; MEC13.3) antibodies (BD Biosciences, SanJose, Calif.) at room temperature for 15 min.

Cells were incubated with biotin selection cocktail for 15 min, and thenincubated with magnetic nanoparticles at room temperature for 10 min.The cups were magnetized, and cells or clumps of cells bound to thewalls of the cup; those not bound (the human cells) were collected intoa separate container. The cells bound to the cups were the mouse cellsthat were discarded. The human cells were suspended in KM and thenplated.

The cells were plated onto culture plastic or on or in hyaluronanhydrogels (some of them supplemented with type III or IV collagen orlaminin) and provided with serum-free KM. For the initial plating, themedium was supplemented with 2-5% FBS (HyClone, Waltham, Mass.). After afew hours, the medium was changed to the serum-free version, and thiswas used for all subsequent medium changes.

For the cultures of xenografted tumors, the human cells were sorted byimmunoselection away from the murine (host) mesenchymal cells and thenwere plated in serum-free KM from the outset.

1.11. Immunocytochemistry and Immunohistochemistry.

For immunofluorescent staining, 5 μm frozen sections or cultured cellswere fixed with 4% paraformaldehyde (PFA) for 20 min at roomtemperature, rinsed with PBS, blocked with 10% goat serum in PBS for 2hours, and rinsed. Fixed cells were incubated with primary antibodies at4° C. for 14 hours, washed, incubated for 1 hour with labeledisotype-specific secondary antibodies, washed, counterstained with4′,6-diamidino-2-phenylindole (DAPI) for visualization of cell nucleiand viewed using Leica DMIRB inverted microscope (Leica, Houston, Tex.)or a Zeiss ApoTome Axiovert 200M (Carl Zeiss Inc, Thornwood, N.Y.).

For immunohistochemistry (IHC), the tissues were fixed in 4% PFAovernight and stored in 70% ethanol. They were embedded in paraffin andcut into 5-μm sections. After deparaffinization, antigen retrieval wasperformed with sodium citrate buffer (pH 6.0) orethylenediaminetetraacetic acid (EDTA) buffer (pH 8.0) in a steamer for20 min. Endogenous peroxidases were blocked by incubation for 15 min in3% H₂O₂. After blocking, primary antibodies reacted against human butnot mouse cells and were applied at 4° C. overnight. M.O.Mimmunodetection kit (Vector Laboratories, Burlingame, Calif.) was usedfor detecting primary mouse anti-human antibodies on mousexenotransplant FL-HCC tumor to avoid the inability of the anti-mousesecondary antibody to endogenous mouse immunoglobulins in the tissue.Sections were incubated for 30 min at room temperature with ImmPRESSperoxidase-micropolymer staining kits and 3,3′-diaminobenzidinesubstrate (Vector Laboratories). Sections were lightly counterstainedwith hematoxylin. Antibodies used are listed in Table 2. Control imagesare given in FIG. 9.

TABLE 2 Antibodies for Immunohistochemistry Man- Reac- Re- AntibodySpecies Isotype ufacture tivity trieval ABCG2 Mouse IgG2a Millipore H CBAFP Mouse IgG2a SIGMA H, D, P CB but not M BMI1 Rabbit IgG Abcam H CBCD44 Mouse IgG2a Abcam H CB CD68 Mouse IgG3 DAKO H CB CK7 Mouse IgG1DAKO H CB CK18 Mouse IgG1 DAKO H CB CK19 Mouse IgG2a Abcam H CBE-cadherin Mouse IgG2b Abcam H CB EpCAM Mouse IgG1 Cell H CB SignalingHepPar-1 Mouse IgG1 DAKO H CB KLF4 Rabbit IgG NOVUS H CB LGR5 Rabbit IgGNOVUS H CB MDR-1 Mouse IgG2a Abcam H EDTA MUC6 Mouse IgG1 Santa Cruz HCB NANOG Mouse IgG1 Cell H EDTA Signaling NCAM Mouse IgG1 DAKO H CB NGN3Rabbit IgG NOVUS H EDTA OCT4 Rabbit IgG Cell H EDTA Signaling PDX1Rabbit IgG NOVUS H CB SALL4 Mouse IgG1 Abcam H EDTA SHH Rabbit IgGMillipore H CB SOX2 Rabbit IgG Cell H CB Signaling SOX9 Rabbit IgG1SIGMA H CB SOX17 Mouse IgG1 Abcam H CB SYNDECAN- Goat IgG R&D H CB 1(HS-PG-1) VCAM-1 Mouse IgG1 Santa Cruz H CB H = human, D = dog, M =mouse, P = pig, R = rat.

1.13. Flow Cytometric Analyses.

The dissociated cells were incubated at 4° C. for 30 min withfluorescein isothiocyanate (FITC)- or biotin-conjugated anti-mouse-MHCclass I (against H-2K^(d)) (clone; 34-1-2S) (eBioscience, San Diego,Calif.) and anti-human antibodies (see Table 3) for cell surfacemarkers. For biotinylated antibody, allophycocyanin (APC)-streptavidin(BD Biosciences, San Jose, Calif.) was used for visualization. The cellswere washed with staining medium before analysis. For the intracellularstaining of LGR5, the cells were incubated with antibodies against thecell surface antigens as usual, and then, were fixed with 4% PFA/PBS at4° C. for 20 min. After washing with staining medium, the cells wereresuspended in permeabilization buffer (PBS with 1% FCS, 0.1% sodiumazide, and 0.1% saponin) with PE-conjugated anti-LGR5 antibody at 4° C.for 30 min. Antibodies used are listed in Table 3. The labeled cellswere washed with permeabilization buffer and then analyzed byFACSCalibur™. (BD Biosciences, San Jose, Calif.).

TABLE 3 Antibodies for Flow Cytometric Analyses Name Clone Host/isotypeManufacture APC-CD13 WM15 Mouse IgG1 eBioscience PE-CD24 ML5 Mouse IgG2aBD Biosciences FITC-CD29 (Integrin β1) TS2/16 Mouse IgG1 eBioscienceAPC-CD44 BJ18 Mouse IgG1 BioLegend FITC-CD49f (Integrin α6) GoH3 RatIgG2a BD Biosciences FITC-CD54 (ICAM) HA54 Mouse IgG1 BioLegend APC-CD56(NCAM) MEM-188 Mouse IgG2a Abcam FITC-CD90 (THY-1) 5E10 Mouse IgG1eBioscience APC-CD117 (c-KIT) YB5.B8 Mouse IgG1 BD BiosciencesAPC-CD133/1 AC133 Mouse IgG1 Miltenyi Biotec APC-CD184 (CXCR4) 12G5Mouse IgG2a eBioscience APC-CD324 (E-cadherin) 67A4 Mouse IgG1 MiltenyiBiotec FITC-CD326 (EpCAM) VU-1D9 Mouse IgG1 Stem Cell TechnologiesPE-TROP-2 77220 Mouse IgG2a R&D PE-LGR5 2A2 Mouse IgG1 Origene APC,allophycocyanin; PE, R-phycoerythrin; FITC, fluorescein isothiocyanate.

1.14. Differentiation Assays.

1×10⁵ hFL-HCC cells, depleted of host mesenchymal cells by magneticsorting, were seeded into each well of a 12-well plate coated with 5μg/cm² hyaluronan and cultured with KM+2% FBS for overnight. After 16-20hours, the cells were incubated for 7 days with either serum-free KM (asthe undifferentiated control) or with serum-free HDM-H, HDM-C or HDM-P.After a total of 7 days culture, cells were harvested for analyses ofgene expression.

1.15. Invasion Assay.

Invasion activity of the cells was analyzed using CultreCoat 96 WellBME-Coated Cell Invasion Optimization Assay Kit (TREVIGEN, Gaithersburg,Md.) according to manufacturer's protocol. The hFL-HCC, depleted of hostmesenchymal cells by magnetic sorting, or the human hepatocellularcarcinoma cell line, Huh7 cells, were cultured with serum-free mediumfor starvation. After 20 hours of serum starvation, cells werecollected. Then 2.5×10⁴ cells were resuspended in 25 μl of serum-freeKubota's Medium (hFL-HCC) or serum-free DMEM (HuH7), and seeded intoeach well of a 96-well culture plates (top chambers). A total of 150 μlof each culture medium+10% FBS were added to the bottom chambers, andcells were cultured for 24 hours. After washing, cells were dissociatedand fluorescently labeled with Cell Dissociation Solution/Calcein AM.After incubation at 37° C. for 1 hour, top chambers were removed and theabsorbance at 485 nm excitation, 520 nm emission was measured.

1.16. Quantitative Reverse Transcription and Polymerase Chain Reactions(qRT-PCR).

Total RNA was extracted from the cells using RNeasy Micro Kit or RNeasyMini Kit (Qiagen GmbH, Valencia, Calif.). First-strand cDNA synthesizedusing the Primescript 1st strand cDNA synthesis kit (Takara, Otsu,Japan) was used as a template for PCR amplification. Quantitativeanalyses of mRNA levels were performed using Power SYBR Green PCR MasterMix with Applied Biosystems 7500 Real-Time PCR System (AppliedBiosystems, Foster City, Calif.). The primers were annealed at 50° C.for 2 min and 95° C. for 10 min, followed by 40 cycles of 95° C. (15 s)and 60° C. (1 min). Expression of glyceraldehyde-3-phosphatedehydrogenase (GAPDH) was used as a control standard. Primer sequencesare listed in Table 4.

TABLE 4 Primers for qRT-PCR Product GenBank Name F/R Primer Sequencelength Accession CD44 F TGCCGCTTTGCAGGTGTAT 66 NM_000610.3 RGGCCTCCGTCCGAGAGA CDH1 F TCACAGTCACTGACACCAACGA 67 NM_004360 RGGCACCTGACCCTTGTACGT CFTR F AAAAGGCCAGCGTTGTCTCC 170 NM_000492.3 RTGAAGCCAGCTCTCTATCCCA KRT7 F TGCTGCCTACATGAGCAAGGT 99 NM_005556.3 RTCTGTCAACTCCGTCTCATTGAG KRT18 F GCCCGCTACGCCCTACA 57 NM_000224.2 RTGACTCAAGGTGCAGCAGGAT KRT19 F CCGCGACTACAGCCACTACT 97 NM_002276.4 RGTCGATCTGCAGGACAATCC LGR5 F GAGGATCTGGTGAGCCTGAGAA 151 NM_001277227.1 RCATAAGTGATGCTGGAGCTGGTAA NANOG F AAATCTAAGAGGTGGCAGAAAAACA 60NM_024865.2 R CTTCTGCGTCACACCATTGC PDX1 F CCCATGGATGAAGTCTACC 262NM_000209.3 R GTCCTCCTCCTTTTTCCAC POU5F1 F GAGAGGCAACCTGGAGAATTTG 58NM_001173531.1 R GATCTGCTGCAGTGTGGGTTT PROM1 F TCCACAGAAATTTACCTACATTGG77 NM_001145851.1 R CAGCAGAGAGCAGATGACCA SOX2 FAAATGGGAGGGGTGCAAAAGAGGAG 112 NM_003106.3 R CAGCTGTCATTTGCTGTGGGTGATGTACSTD1 F GACTTTTGCCGCAGCTCAGGAAG 135 NM_002354.1 RGCCAGCTTTGAGCAAATGACAGTATTTTG GAPDH F AAGGTGAAGGTCGGAGTCAA 108NM_002046.3 R AATGAAGGGGTCATTGATGG

1.17. Cell Proliferation and Chemo-Resistance Assays.

For cell proliferation assays, 3×10⁴ hFL-HCC cells of TU-2010 weredepleted of host mesenchymal cells by magnetic sorting ofxenotransplantable tumor cell suspension and then were seeded into eachwell of a 96-well plate and cultured overnight with Kubota's Medium+5%FBS. After 16-20 hours, the specific hedgehog inhibitor Vismodegib(GDC-0449) (Selleckchem Bio, Houston, Tex.) or the histone deacetylase(HDAC) inhibitors, suberic bis-hydroxamic acid (SBHA) or suberoylanilidehydroxamic acid (SAHA) (SIGMA, St. Louis, Mo.) were added for 3 days.Cell proliferation was evaluated using the Cell Proliferation ReagentWST-1 (Roche Applied Science, Mannheim, Germany). After incubation at37° C. for 2 hours, the absorbance at 450 nm was measured.

1.18. Spheroid Formation Assays.

For spheroid formation assays, 1×10⁴ hFL-HCC cells of TU-2010, depletedof host mesenchymal cells by magnetic sorting, were seeded into eachwell of a 6-well plate coated with Ultra-Low Attachment surface(Corning, Lowell, Mass.) and cultured with serum-free Kubota's Medium inthe presence (or absence) of the specific hedgehog inhibitor Vismodegib(GDC-0449) or the HDAC inhibitors, SBHA or SAHA. For secondary spheroidformation assays, the 1st spheroids were collected, subsequentlydissociated with NeuroCult Chemical Dissociation Kit (STEMCELLTechnologies). Cell suspension was centrifuged at 700 rpm 10 min andresuspended with KM. After 2 weeks, the number of spheroids (100 μm>)were counted.

1.19. Transmission Electron Microscopy.

The hFL-HCC spheroids of Tu-210 were fixed with 3% glutaraldehyde in0.15M sodium phosphate buffer, pH 7.4, for 1 hour at room temperatureand stored at 4° C. until processed. Following three rinses with 0.15 Msodium phosphate buffer, pH 7.4, the samples were post-fixed for 1 hourwith 1% osmium tetroxide/1.25% potassium ferrocyanide/0.15M sodiumphosphate buffer, pH 7.4, followed by rinses in deionized water. Thespheroids were dehydrated using increasing concentrations of ethanol(30%, 50%, 75%, 100%, 100%, 10 min each) and 2 changes of propyleneoxide (15 min each). Following infiltration overnight in a 1:1 mixtureof propylene oxide/Polybed 812 epoxy resin (Polysciences, Inc.) and 24hours in 100% resin for 24 hours, the spheroids were embedded in freshPolybed 812 epoxy resin. The spheroids were sectioned transversely at 70nm using a diamond knife and a Leica Ultracut UCT microtome (LeicaMicrosystems, Wetzlar, Germany). Ultrathin sections were mounted on 200mesh copper grids and stained with 4% aqueous uranyl acetate andReynolds' lead citrate. The grids were observed at 80 kV using a LEOEM910 transmission electron microscope (Carl Zeiss SMT, LLC). Digitalimages were taken using a Gatan Orius SC 1000 CCD Camera withDigitalMicrograph 3.11.0 software (Gatan, Inc., Pleasantan, Calif.).

1.20. RNA-Sequencing and Gene Expression Analysis.

RNA was purified using Qiagen RNeasy Kit from human adult hepatocytes,hepatoblasts, hepatic stem cells, and biliary tree stem cells, each fromthree different donors, as well as four FL-HCC tumor samples of passagedTU-2010. In addition, RNA was purified from three cancer stem cellpopulations of the liver from tumors that are presumptive transformantsof: (1) HpSCs, giving rise to hepatocellular carcinoma (HCC); (2) latestage (EpCAM⁺) BTSCs, giving rise to cholangiocarcinoma (CCA); and (3)primitive BTSCs (EpCAM⁻, CD44⁺), giving rise to fibrolamellar carcinoma(FLC). RNA integrity (RIN) analysis was performed using an Agilent 2000Bioanalyzer. cDNA libraries were generated using the Illumina TruSeqStranded mRNA preparation kit and sequenced on the Illumina HiSeq 2500platform. Two samples were sequenced per lane, occupying a total of 8lanes for all of the samples (one flow cell). Quality control analysiswas completed using FastQC, mapping of sequence reads to the humangenome (hg19) was performed with MapSplice2 using default parameters,transcript quantification was carried out by RSEM analysis, and DESeqwas used to normalize gene expression and identify differentiallyexpressed genes. MapSplice2 was also used to detect candidate fusiontranscripts. Fusion calls were based on the depth and complexity ofreads spanning candidate fusion junctions. Gene expression profiles werecompared using Pearson's correlation analysis and hierarchicalclustering was performed in R. Pathway enrichment analysis was performedwith the Ingenuity Pathway Analysis (IPA) software.

Genes were determined to be differentially expressed between cancertypes if they had >50 average normalized counts in at least one tumortype, exhibited >2-fold change, and had an adjusted p-value <0.05.Within the 163 genes found to be significantly differentially expressedin FLC compared to HCC or CCA, 16 genes were further identified forwhich the expression level in all FLC samples was greater than theexpression level in 95% of HCC and 95% of CCA samples. The expression ofthese 16 genes was compared between a FLC patient-derived xenograft(PDX) model and normal biliary tree stem cells that were previouslysequenced as described above. For RNA-seq analysis across 24 differenttumor types, pre-processed RNA-seq data were downloaded from TCGA andplotted in R.

1.21. Normal Human Biliary Tree Stem Cells (hBTSCs).

The biliary tree contains stem cell niches, peribiliary glands (PBGs),mucinous glands scattered as intramural PBGs within the walls of thebile ducts and also found as extramural PBGs that are tethered to thebile ducts. The phenotypes of the cells within the PBGs can berelatively homogeneous in some sites (e.g. hepato-pancreatic common ductand intrahepatic, large bile ducts) and quite heterogeneous in othersites (e.g. cystic duct, common duct, hepatic duct). The pattern ofphenotypic traits of the PBG cells was found to implicate maturationallineages in a radial axis from the fibromuscular layer within the ductwalls to the lumen of the bile ducts and in a proximal(duodenum)-to-distal axis from the duodenum to either liver or pancreas.

The PBGs deepest within the bile ducts and near the fibromuscular layercontain the most primitive stem cells, those that co-expresstranscription factors for both liver and pancreas (e.g. SOX 17, PDX1)and that also co-express multiple pluripotency genes (e.g. OCT4, SOX2,KLF4, NANOG). These cells do not express epithelial cell adhesionmolecule (EpCAM) or even LGR5. These are referred to as stage 1 biliarytree stem cells (hBTSCs) They transition to PBGs with cells expressingLGR5 (stage 2 hBTSCs) and then to ones positive for both LGR5 and EpCAM(stage 3 hBTSCs) found at levels that are intermediate within the bileducts. With transition to the luminal surface of the ducts, there isacquisition of cells with mature phenotypic markers. If the ducts arenear or within the liver, the mature markers are those for liver; ifthey are within the hepato-pancreatic common duct, the mature markersare pancreatic.

1.22. Cultures of Normal hBTSCs.

The stage 1 hBTSCs have not yet been successfully cultured under theconditions tested. These have yet to be observed in culture under theconditions used. Two stages of hBTSCs that have been observed under theconditions used are stage 2 and 3 hBTSCs. See FIGS. 18 to 20. The stage3-hBTSC colonies strongly express both LGR5 and EpCAM in every cell andform colonies of relatively uniform, cuboidal shaped cells that aretightly bound to each other. They are distinct from stage 2-hBTSCs thatare undulating, swirling cells that can form extensions, are highlymotile and have variable connections with neighboring cells. Thesecolonies strongly express LGR5 throughout all of the cells, but aredevoid of EpCAM expression on the interior of the colonies and yetexpress it at their edges in cells that are slightly larger and moredifferentiated. Treatment of the stage 2 hBTSC colonies with any ofseveral different growth factors (e.g. EGF, HGF) or with laminin resultsin rapid transition to stage 3 hBTSCs with activation of expression ofEpCAM throughout the colony. The net results indicate that the stage2-hBTSCs [LGR5+, EpCAM-negative cells], are precursors of the stage3-hBTSCs (LGR5+, EpCAM+].

A summary of phenotypic traits of biliary tree stem cells versus hepaticstem cells is given in Table 5. They indicate that the lineages ofbiliary tree stem cells are precursors of hepatic and pancreatic stemcells and are assumed to contribute to organogenesis of liver versuspancreas. A chart of the lineage stages identified is given in FIG. 20.The cells in the hFL-HCC tumor line, TU-2010, are most closely similarto the stage 2-hBTSCs.

TAABLE 5 Phenotypic Profile of Normal Stem Cells in Liver and BiliaryTree and of Pancreatic Committed Progenitors Versus Human FibrolamellarHepatocellular Carcinoma Cells (hFL-HCC) of the TU-2010 tumor lineLiver 

 Pancreas Committed hHpSCs Progenitors (in Canals hBTSC Subpopulations(in Pancreatic hFL-HCC Cells Property of Hering) (in peribiliary glands)Duct Glands) of TU-2010 Endodermal SOX 9, SOX 9, SOX 9, SOX 9, PDX1,LGR5 SOX 9, SOX17, markers LGR5, SOX17 SOX17, PDX1 PDX1, LGR5, HNF4A,LGR5 PDX1 LGR5 FOXL1, HNF4A FOXL1 FOXL1 FOXL1 FOXL1 HNF4A HNF4A HNF4AMarkers of CK 8 and 18, CK 7 and 19, E-cadherin Epithelia Cell NCAM,NCAM, NCAM NCAM, EpCAM+ NCAM, VCAM, Adhesion EpCAM + EpCAM EpCAM EpCAMEpCAM ± Molecules ITGB1 (CD29) + − + (negligible) ITGA6 (CD49f), ITGB4,ITGA6 (CD49f), ITGB1 (CD29) ITGB4, ITGB1 (CD29) Pluri- OCT4, KLF4/KLF5,NANOG, None KLF4/KLF5, potency SALL4, OCT4, SALL4, TROP-2, OCT4, GenesNANOG BMI1 NANOG, SALL4,BMI1 Other Stem CXCR4, CXCR4, CD133; and NoneCD133, Sonic Cell CD133, Hedgehog proteins (Indian Hedghog, ALDH MarkersHedgehog and Sonic), ALDH proteins (Indian, Sonic), ALDH ProteinLaminin, Laminin, Oncostatin M Fetal islets: Laminin, Matrix type IIIreceptor collagens IV, V, Oncostatin M components collagen, VI, Laminin,receptor, CD68 Receptors Oncostatin Nidogen, elastin, M receptor fetalacinar cells: fibrillar collagens, fibronectin GAGs/PGs MinimallyHyaluronans, CD44. Hyaluronans, Hyaluronans, sulfated CD44, fetal isletsCD44, CS-PGs, have syndecans Syndecan-1 (HS- Hyaluronans (HS-PG-1 and3), PG-1) glypicans, fetal acinar cells have CS-PGs Liver- Albumin +/−,KRT7, HNF4A None AFP-, HNF4A specific AFP-, HepPar-1+; traits HNF4A,KRT7 HepPar1+, KRT7, DLK1 Pancreatic- PDX1 PDX1 PDX1, PDX1, NGN3, PDX1,KRT20, specific ISL1, MAFA, MUC6, NGN3 traits NGN3 Nkx6, PTF1a, GLUT2Multidrug MDR-1, ABCG2 None MDR-1 resistance ABCG2 genes

Experimental Results.

Endodermal Stem/Progenitor Markers were Expressed in hFL-HCCs.

Sections from original blocks of 9 hFL-HCC tumors from Memorial SloanKettering Cancer Center (MSKCC) were subjected to IHC assays (FIGS. 1and 7). All sections assayed were positive for HepPar-1 and SHH.Positive expression was also observed in 7/9 for SOX9 and PDX1 and 4/9for BMI1.

Tissue microarrays (FIG. 8) from Memorial Sloan Kettering Cancer Center(MSKCC) provided additional evidence from primary tumors. Whereasstem/progenitor traits were not observed in any of the 19 normal livers,all hFL-HCCs were positive for multiple stemness markers: SOX9 (12/18),SOX17 (8/18), OCT4 (7/18), SALL4 (3/6), SHH (18/18), and PDX1 (13/18).

A Transplantable hFL-HCC Tumor Line, TU-2010, was EstablishedSuccessfully by Use of Culture-Selected Endodermal Stem/Progenitors.

A young, male patient was diagnosed with FL-HCC and was subjected toliver surgery and chemotherapies, all of which proved unsuccessful(Table 1). Within 2 years, the tumor had metastasized and generatedascites tumor cells. Approximately 5 liters of ascites fluid wereremoved from the patient. Cells from one liter were immediatelytransplanted into immune-compromised mice, but no tumors formed. Cellsfrom the remaining 4 liters were subjected to culture selection inKubota's Medium (KM) for endodermal stem/progenitors, and 2×10⁷ cellswere transplanted into nod scid gamma (NSG) mice. Tumor formationoccurred after >6 months.

Passaging of hFL-HCC of TU-2010 was Stabilized by Supplements andOccurred with Different Kinetics in Subcutaneous Versus IntraperitonealSites.

Tumors were passaged every 3-5 months and stabilized at about 3 monthswith transplantation of about 10⁶ cells in KM supplemented with 1 mg/mlhyaluronans and with 50 ng/ml each of hepatocyte growth factor (HGF) andvascular endothelial cell growth factor (VEGF) (FIG. 2). Thepassageable, subcutaneous tumors were nodular and difficult to mince. Iftransplanted intraperitoneally (FIGS. 2e and 2h ), ascites tumorsformed, requiring passaging every about 8 weeks and giving rise tonodules on serosal surfaces throughout the peritoneum and on liver andpancreas.

Histology of Xenografted Tumor Cells of TU-2010 Matched that of theOriginal Tumor.

The histology of the original tumor (FIG. 2a-2b ) and of the ascitestumor cells (FIG. 9a ) versus those of xenografts (FIG. 3d-3g and FIG.9b ) revealed differences between the tumor centers (FIGS. 2d 1 and 2 f1) and their perimeters (FIGS. 2d 2 and 2 f 2), sites at which tumorsinterfaced with host tissues. Tumor centers demonstrated histologysimilar to that of the original tumor with large polygonal cells,abundant eosinophilic cytoplasm, large, vesiculated nuclei and largenucleoli. By contrast, the histology at tumor perimeters comprisedincomplete ductular structures with partially stabilized lumens and withfeatures similar to those of intrahepatic, mixed-typecholangiocarcinomas (CCs) with ductular areas.

The FL-HCC Tumors of TU-2010 were Comprised Primarily of Host Cells.

Mesenchymal cells within tumors were a mix of precursors to stellatecells (desmin⁺, alpha-smooth muscle actin⁺) and endothelia (CD31⁺) andcomprised, on average, 55-70% of cell suspensions from subcutaneoustumors and >95% of those from intraperitoneal tumors (FIG. 2g ).Enrichment of hFL-HCCs to ≧95% (FIG. 2h ) was achieved by negativesorting using magnetic bead immunoselection to eliminate murine cells,ones positive for H-2K^(d) (FIG. 2g ). Tumors contained, on average,about 8×10⁶ hFL-HCC cells/gm of tumor.

Expression of Stem/Progenitor Markers in TU-2010 Cells was Confirmed byFlow Cytometry.

Immunoselected hFL-HCCs, depleted of murine cells, were characterized bymultiparametric flow cytometry (FIG. 3a-3b ). The majority of cells werepositive for LGR5 (68.9%) and CD44 (61.4%); a significant percentagewere positive for CD29 (43.7%), CD24 (32.9%), CD49f (25.4%), CD13(12.5%), E-cadherin (12.0%), c-KIT (12.0%) and oncostatin Mreceptor-OSMR (10.7%). A low but consistent percentage of cells werepositive for NCAM (3.7%), EpCAM (4.3%), CXCR4 (4.8%), CD133 (2.3%),TROP-2 (1.4%) and ICAM (0.5%). A small percentage (1.1%) of LGR5+ cellswere positive for EpCAM.

The hFL-HCCs of TU-2010 were Rich in Cancer Stem Cells (CSCs) asIndicated Functionally by Limiting Dilution Tumorigenicity Assays.

Cell suspensions were depleted of murine cells and transplantedsubcutaneously into NSG mice in tumorigenicity assays from 100 to 10⁶cells and monitored for up to 8 months for tumors. Transplantation of10⁵ or more cells resulted in 100% of the mice developing tumors withinabout 3 months; 10³-10⁴ cells within 5-6 months; and 100 cells in allmice but requiring up to 9 months (FIG. 2i ).

Xenografted hFL-HCCs of TU-2010 Expressed Endodermal Stem/ProgenitorMarkers that Collectively Suggest Derivation from Biliary Tree StemCells (hBTSCs).

Sections of xenografted hFL-HCCs were subjected to IHC assays (FIG.3d-3g and FIG. 9b ) and the findings compared to those from originaltumor cells (FIG. 9a ). The hFL-HCCs were positive for CD68, apreviously observed feature of hFL-HCCs; stem/progenitor markers(SOX17,SOX9,LGR5,SHH,NCAM,BMI1); pluripotency traits (NANOG, OCT4, KLF4,SOX2, SALL4); some hepatic markers (HepPar-1,CK7,CK19,CK18); andpancreatic markers (PDX1). They were essentially negative for albumin,alpha-fetoprotein (AFP), MUC6, and weakly positive for EpCAM (FIG. 3f ).The markers were suggestive of highly aggressive tumors.

Other markers (FIG. 9b ) in TU-2010 cells included some multidrugresistance genes and matrix components that facilitate cell survival andgrowth: E-cadherin; syndecan-1 (HS-PG1); hyaluronan receptors (CD44);and vascular cell adhesion molecule-1 (VCAM-1). The cells did notexpress hemopoietic (CD34, CD45), stellate (CD146) or endothelial cellantigens (CD31). Negative controls are shown in FIG. 9b . A summary ofin situ analyses of hFL-HCC cells as well as normal human hepatic stemcells (hHpSCs), hepatoblasts (hHBs) and hBTSCs is provided in Table 5and in FIGS. 18-20. The hFL-HCC tumor most closely resembledstage-2-hBTSCs (FIG. 20).

The hFL-HCC Cells of TU-2010 in Culture Behaved Similarly to NormalhBTSCs.

That hFL-HCC cells derive from stage-2-hBTSCs was supported further bytheir behavior in monolayer (FIGS. 4 and 10) and in spheroid cultures(FIGS. 5, 11 and 12). In monolayer cultures, the original hFL-HCCsattached and formed star-like cells (FIGS. 4a and 10), but transitionedrapidly into cells loosely attached and connected to floating cellchains (FIG. 4b ), with cells bound to each other via E-cadherinlinkages.

If plated overnight with 2-5% fetal bovine serum (FBS) and then switchedto serum-free KM (FIG. 4c-4d ) or if depleted of murine cells and platedin KM (FIG. 4e ), hFL-HCC colonies remained attached, spread and formedcolonies strongly expressing pluripotency genes (e.g., NANOG) andstem/progenitor markers (e.g., CD44, LGR5). In vitro invasion assays(FIG. 4f ) indicated the invasive properties of hFL-HCCs were greaterthan Huh7, a human liver cancer cell line, correlating with the invasivepotential of hFL-HCCs in vivo, especially with intraperitonealtransplants.

Colony morphologies of hFL-HCCs of TU-2010 were similar to those ofstage-2-hBTSCs (FIGS. 18 and 19)—motile cells formed partially ductularstructures and were negative for EpCAM in colony centers buttransitioned to weak EpCAM expression at colony perimeters.

Assays for drug effects on monolayer cultures (FIG. 4g ) of TU-2010cells indicated that the hedgehog signaling pathway inhibitor, GDC-0449,at 4 μM and especially at 20 μM, and two histone deacetylase (HDAC)inhibitors, SBHA (Suberic bis-hydroxamic acid) at 10 μM and 50 μM andSAHA (suberoylanilide hydroxamic acid) at 2 μM and 10 μM, stronglyinhibited hFL-HCC cell growth.

TU-2010 Spheroids, Indicative of Self-Replicative Ability and,Therefore, of CSCs, Formed in KM.

Spheroid formation required that serum-free KM be used throughout,including for plating of cells; spheroids proved able to be passaged formonths (FIG. 5a ). Transmission electron microscopy (TEM) of spheroidsin KM (FIGS. 5b , 11 and 12) revealed tumor cells with microvilli attheir apical poles, indicating ability to polarize. Cells were rich inrough endoplasmic reticulum (RER) and Golgi (G), with numerous secretoryvesicles containing electron-dense granules typically associated withneuroendocrine traits (e.g. chromogranin) such as occurs in pancreatictumors. Nuclei contained dispersed chromatin and large nucleoli,implicating high production of secretory proteins. Cells were rich inpleomorphic, irregularly-shaped, non-lucent mitochondria with irregulardisorganized cristae. Cells in spheroids proved even more sensitive toinhibition by drugs (SAHA, SBHA, GDC-0449) than in monolayers (FIG. 5d).

Differentiation Media, Used to Lineage Restrict Normal hBTSCs to AdultFates, Caused hFL-HCCs of TU-2010 to Lose Stemness Traits.

Serum-free, hormonally defined media (HDM), established previously forlineage restriction of hBTSCs to hepatocytes (HDM-H), cholangiocytes(HDM-C) or pancreatic islets (HDM-P), were used to differentiatehFL-HCCs. Cells were monitored for morphological (FIG. 5e ) and IHCchanges (FIG. 5f ) and were assayed by qRT-PCR (FIG. 5g ) for stemness(e.g., NANOG, POU5F1, SOX2) and mature markers (e.g., CFTR). Peak levelsof stemness traits occurred in KM, whereas those markers weresignificantly suppressed in all three HDM. In HDM-C, there was anincrease in CFTR mRNA and protein (FIG. 5f ). CFTR is found in normalstem cells but increases in levels during maturation to cholangiocytes.Higher levels of differentiation were blocked by hFL-HCCs production ofmatrix-degrading factors.

Transcriptomic Analyses Revealed that hFL-HCCs of TU-2010 Most CloselyResemble hBTSCs.

Paired-end high-throughput RNA sequencing was conducted in purifiedpopulations of adult human hepatocytes (hAHEPs), hHBs, hHpSCs, andhBTSCs, each from three different donors, as well as four hFL-HCCs fromdifferent passaged lines of the transplantable tumor (FIG. 6). Anaverage of about 200 million paired-end reads per sample were obtained,of which an average of about 87% mapped uniquely to the human genome.Gene expression profiles were strongly correlated among samples withineach category (average Pearson's r²=0.98 for the hFL-HCC preparations;0.88 for hHBs and hBTSCs; and 0.81 for hHpSCs) (FIG. 6a ). The highcorrelation among the hFL-HCC samples indicated remarkable stability ofgene expression throughout four years of passaging in mice.Cross-category comparisons revealed that gene expression profiles ofhFL-HCCs were most strongly correlated with those of hBTSCs (FIG. 6a ).This finding was further supported by results of hierarchical clusteringanalyses, showing that hFL-HCCs are more closely related to hBTSCs thanhHpSCs, hHBs, or hAHEPs (FIGS. 6b-6c and 13).

Unique features of hFL-HCCs of TU-2010 (FIG. 6d ) included highexpression of AGR2; DCLK1; and KRT20, all found in endodermal cancers,particularly of intestine; KLF4/5, critical regulators of stemness; andAHR, shown to trigger malignant transformation of stem cells uponbinding to dioxins and related agonists. Interestingly, HDAC9, which ismost highly expressed in hBTSCs, is missing altogether in hFL-HCCs andhas been linked to tumor suppressive activity through effects on p53.

Expression data are shown for representative stem/progenitor,hepatocytic, biliary, and pancreatic genes (FIG. 14), components of thehedgehog signaling pathway (FIG. 15), and HDAC genes (FIG. 16). Resultsof pathway enrichment analysis for genes differentially expressed inhFL-HCCs compared to hHpSCs or hBTSCs are shown in FIG. 17.

Finally, RNA-seq data were further analyzed using MapSplice2 anddetected with high confidence a recurrent fusion transcript unique tohFL-HCCs, DNAJB1-PRKACA, for which Sashimi plots are shown in FIG. 6e .This chimera was identified previously in hFL-HCC tumors, and wasdemonstrate to be uniquely expressed in hFL-HCCs and not in normal stemcells or adult hepatocytes.

Transcriptomic Analyses Revealed Unique Molecular Signature of hFL-HCCsand Candidate Therapeutic Targets.

To further examine the molecular characteristics of hFL-HCCs, the TheCancer Genome Atlas (TCGA) liver cancer database was mined for hFL-HCCs.Based on the presence of DNAJB1-PRKACA and/or classic histologicalfeatures, 7 hFL-HCC samples were identified, four of which wereincorrectly annotated by TCGA as HCCs. The gene expression profiles ofthese 7 hFL-HCCs clustered most closely with each other and were clearlydistinct from HCCs (n=262) and CCAs (n=36). Expression levels ofpreviously suggested RNA markers of hFL-HCC were analyzed (e.g. AGR2,KRT7, and NTS), and it was found that, with the exception of PCSK1 andDNAJB1-PRKACA, none appear to uniquely mark hFL-HCC relative to HCCs,CCAs, normal livers (n=50), or normal cholangiocytes (n=9) and thus maynot be clinically actionable (Data not shown).

Therefore, a comprehensive transcriptomic analysis was performed andidentified a suite of 165 genes that were significantly altered inhFL-HCCs relative to HCCs and CCAs (FIG. 21). Furthermore, all of thehFL-HCC samples exhibited greater expression than 95% of the HCC and CCAsamples for the 16 genes (FIG. 22). The elevated expression of the 16genes, were further validated in an independent hFL-HCC sample (set ofhFL-HCC samples (originally described by Dr. Sanford Simon, RockefellerUniversity, NYC) and non-tumor liver and non-tumor cholangiocytes (FIG.23). Among these, the following 7 genes were the most unique to FLC:PCSK1, CA12, NOVA1, SLC16A14, TNRC6C, TMEM163, and RPS6KA2 (FIG. 22).None of these except PCSK1 have been reported previously as hFL-HCCmarkers. In addition 8 genes (C10orf128, OAT, PAK3, PCSK1, PHACTR2,SLC16A14, TMEM163, and TNR6C) have a greater average of expression inhFL-HCCs as compared to 23 other tumor types from different tissue (FIG.25). To determine which, if any, of these genes are the strongestcandidates for drivers of FLC tumor progression, expression levels inthe hFL-HCC tumor model was compared with its presumptive normalcounterpart, BTSCs. Surprisingly, all 7 were elevated in the tumor modelrelative to hBTSCs, and 5 genes were significantly altered (FIG. 24). Inaddition, genes that are differentially expressed in hFL-HCCs relativeto hBTSCs are significantly enriched for predicted target sites ofseveral microRNAs (miRNAs), including miR-10b, which has been implicatedin tumorigenesis and the maintenance of CSCs. Quantitative PCR analysisrevealed that miR-10b was significantly up-regulated (˜17-fold, P=0.03)in hFL-HCCs compared to BTSCs whereas a control miRNA, one notimplicated in cancer stem cell maintenance, miR-33a, was unaltered (Datanot shown). Together, these data suggest novel markers and drivers ofcancer stem cells in hFL-HCCs.

Gene Ontology Molecular Function Analysis to Identify Network HubProteins for Targeted hFL-HCC Therapeutics

Identifying the protein networks involved in the molecular signature ofhFL-HCCs could not only help it providing potential mechanisms of actionbut could also help to identify additional candidate therapeutictargets. It is contemplated that by controlling “upstream” or“downstream” targets of the genes of the hFL-HCC signature, one may beable to better treat hFL-HCC.

Gene ontology molecular function analyses were performed and revealedthat the 165 hFL-HCC specific genes are enriched in kinase activity,growth factor binding, and cAMP (cyclic adenosine monophosphate)binding, suggesting potential mechanisms of action. FIG. 26. Inaddition, Kinase Enrichment Analysis results of the 165 hFL-HCC genesignature showed enrichment in substrate targets of Protein kinase Acatalytic subunit alpha (PRKACA). These substrate targets include, forexample, tyrosine kinase with immunoglobulin-like and EGF-like domains 1(TIE1); G protein-coupled receptor kinase 1 (GRK1); kinase insert domainreceptor (KDR); sarcoma (SRC) (gene); casein kinase 2 subunit alpha(CSNK2A2); protein kinase c alpha (PRKCA); mitogen-activated proteinkinase 14 (MAPK14); cyclin-dependent kinase 1 (CDK1); and epidermalgrowth factor receptor (EGFR). FIG. 27.

Protein-Protein Interaction (PPI) Hub Protein analysis showed PRKACA andsarcoma (SRC) gene may function as network hubs in hFL-HCCs. Additionalproteins in these hubs include, for example, tyrosine kinase withimmunoglobulin-like and EGF-like domains 1 (TIE1); G protein-coupledreceptor kinase 1 (GRK1); catenin beta-1 (CTNNB1); caveolin-1 (CAV1);protein kinase c alpha (PRKCA); protein tyrosine phosphotase,non-receptor type 11 (PTPN11); Src homology 2 domain containingtransforming protein 1 (SHC1); phospholipase C, gamma 1 (PLCG1); V-crkavian sarcoma virus CT10 oncogene homolog (CRK); andphosphoinositide-3-kinase, regulatory subunit 1 (PIK3R1). FIG. 28

DISCUSSION

Phenotypic properties of hFL-HCCs, rare liver cancers, derive in partfrom their richness in CSCs (over 60% in the transplantable tumor line)and their origins from hBTSCs, precursors to liver and pancreas. Thesefindings provide clarifications for hFL-HCCs' hepatic, cholangiocytic,and endocrine markers, as well as intestinal traits, and for why the5-year survival is only 45%, the overall mortality is 60%, and half thepatients have metastases at the point of diagnosis.

The hFL-HCCs have increased in frequency from an unrecognized livercancer in the 1970s to about 5% of all liver cancers today. As yet,there is no explanation for this increase. Nor is it understood whypatients are primarily children to young adults, and more rarely,middle-aged adults, with no prior history of liver disease. The findingsof remarkably high levels of AHR receptors in hFL-HCCs and in hBTSCs, incombination with prior report that dioxins preferentially affect stemcells, provides clues about the possible aetiological factors ofhFL-HCCs. AHR agonists emerged as environmental factors from the plasticindustries since World War II, correlating with increased incidence ofhFL-HCCs.

The properties of hFL-HCCs implicate origins from biliary tree stemcells, precursors to liver and pancreas and located in peribiliaryglands (PBGs) and in crypts at the base of villi within gallbladders.Lineage tracing studies in mammals and zebra fish indicate that thebiliary tree is a major reservoir of stem/progenitors contributing toliver organogenesis and, as determined recently, pancreaticorganogenesis.

Early stages of malignant transformation of hBTSCs within PBGs have beendescribed. PBGs replete with EpCAM-negative hBTSCs are found in PBGsnear the fibromuscular layers throughout the biliary tree, including inthe large intrahepatic bile ducts.

IHCs and histology provided evidence for the relationship of hFL-HCCs toendodermal stem/progenitors. Histology demonstrated the typical bands ofstroma surrounding clumps of large tumor cells having prominent nucleiand aberrations in mitochondria. Co-expression was found forstem/progenitor markers (NANOG, OCT4, SALL4, SHH) and endodermaltranscription factors (SOX9, SOX17). The hFL-HCCs expressed some hepatictraits (e.g., HNF4, HepPar-1), and the remainder expressed pancreatictraits (e.g., PDX1, PCSK1) or both.

More extensive analyses were made possible by establishment of thefirst-ever model of hFL-HCCs, TU-2010, a transplantable tumor linemaintained in NSG mice. Prior efforts to produce hFL-HCC tumor lines (orcell lines) failed, including those with the ascites tumor cells able togenerate a tumor line for these studies. Success proved dependent onculture selection in Kubota's Medium, a serum-free medium designed forendodermal stem/progenitors and not permissive for survival of latermaturational lineage stages. The speed of passaging was enhanced withsupplements, particularly hyaluronans, HGF and VEGF.

Striking features of the transplantable tumor line, TU-2010, were itsdesmoplastic traits. Although high levels of tumor stroma occur in HCCsand in CCAs, the transplantable hFL-HCC line, TU-2010 generatedsubcutaneous tumors comprised of 55-70% host stroma and intraperitonealones with more than 95% host stroma. Immunoselection to remove hostcells resulted in tumor cells readily cultured as spheroids and withphenotypic traits consistently expressed even after years of passagingin NSG mice (FIG. 6a ). Tumor stroma produced paracrine signals (matrixand soluble signals), which are important in tumor progression andmetastasis.

Phenotypic analyses of hFL-HCCs from TU-2010, after removal of hostcells, were consistent with those from primary tumors and indicated arelationship to hBTSCs. The tumor line, TU-2010, is strikingly rich incancer stem cells (CSCs; >65% CSCs based on proportion of LGR5+ cells),a unique finding given that the average percentage of CSCs in HCCs isabout 0.5-3%, and that in CCAs is about 10-20%. The richness of CSCs inhFL-HCCs was demonstrated functionally by their ability to form tumorsin 100% of the mice with as few as 100 cells and by the relative easewith which they formed spheroids or organoids in culture.

The TEM studies on the spheroids from TU-2010 revealed many noteworthyfeatures, but perhaps the most striking were the electron-dense granulesand the extraordinary numbers of mitochondria with abnormal cristae, acondition typical of certain cancers. This suggests that themitochondria generated ATP by oxidative phosphorylation and made thecells tolerant of hypoxia. An oncocytic condition with such pleomorphicmitochondria is not known to be associated with HCCs but with pancreaticcancers. The secretory granules could contain factors responsible forthe ability of hFL-HCCs to dissolve every type of matrix tested assubstratum.

The strongest evidence of hBTSCs as the origins of hFL-HCCs derives fromRNA-seq studies, which includes analyses of genes across successivelineage stages from hBTSCs to hHpSCs to hHBs to adult hepatocytes. Theglobal transcriptome-wide analyses indicate that hFL-HCCs from TU-2010much more closely resemble hBTSCs than the other lineage stagesanalyzed. Also, the RNA-seq analyses independently confirmed thathFL-HCCs uniquely express the DNAJB1-PRKACA chimera, a fusion genecoupling the catalytic site of protein kinase A (PKA) and a heat shockprotein, resulting in stable activation of PKA.

Genetic analyses have identified unique patterns of claudins, tricellin,CD68, and other biomarkers, ones distinct from those in other livercancers. Earlier studies also indicated that hFL-HCCs have MosaicG-protein alpha-subunit (GNAS)-activating mutations, characterized bySTAT3 activation, EGF receptor levels higher than in other types ofhepatic tumors, and no K-RAS mutations.

The resistance of hFL-HCCs to chemotherapies is predictable, given thecells' expression of multidrug resistance genes. Their renownedaggressiveness in patients and in immune-compromised hosts correlateswith expression of multiple genes, including adhesion molecules(E-cadherin, VCAM-1), matrix receptors (CD44), and syndecan-1 (HS-PG),known for binding FGFs, VEGFs, and other growth factors and presentingthem as potent mitogens.

The findings that HDAC and hedgehog inhibitors are potent suppressors ofgrowth and survival of hFL-HCCs from TU-2010 indicate new therapeuticoptions. Similar effects were previously observed with hedgehoginhibitors on normal stem cells, a finding complemented by parallels inexpression of hedgehog genes in hFL-HCC versus hBTSCs. By contrast,expression patterns of HDAC genes are distinct in hFL-HCCs versus otherparenchymal lineage stages. An intriguing finding is the complete lossof HDAC9 in hFL-HCC.

The TU-2010 tumor's richness in CSCs, the probable origins from biliarytree stem cells, the finding that AHR agonists could be etiologicalfactors in the cancer, and the transplantable tumor line describedherein offer novel diagnostic and therapeutic options, ones much neededfor this devastating liver cancer.

The discovery of unique molecular signatures for hFL-HCCs will aid inearly detection and identification of appropriate therapeutic regimens.In addition to conventional treatment options, these findings suggestseveral novel candidate therapeutic options. In particular, smallmolecule inhibitors of CA12 may be used to suppress cancer growth andare currently in preclinical development. In addition, because aberrantmiR-10b regulatory activity may contribute to hFL-HCCs pathogenesis,miR-10b and its target genes may be candidate therapeutic targets.Locked nucleic acids (LNAs) may also be useful for potent inhibition ofmiRNAs and other genes. LNAs have been developed for both research andtherapeutic use. Finally, immunotherapies may be novel candidatetherapeutics for treating hFL-HCCs.

In the foregoing description, it will be readily apparent to one skilledin the art that varying substitutions and modifications may be made tothe invention disclosed herein without departing from the scope andspirit of the invention. The invention illustratively described hereinsuitably may be practiced in the absence of any element or elements,limitation or limitations, which is not specifically disclosed herein.The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention. Thus, it should be understood that although the presentinvention has been illustrated by specific embodiments and optionalfeatures, modification and/or variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scopes ofthis invention.

Para. A. A transplantable tumor line of human fibrolamellarhepatocellular carcinoma (hFL-HCC) cells maintained in a non-humananimal.

Para. B. The transplantable tumor line of Para A, wherein the non-humananimal is immunocompromised.

Para. C. The transplantable tumor line of Paras. A or B, wherein thenon-human animal is a mouse.

Para. D. The transplantable tumor line of any one Paras. A-C, whereinthe non-human animal is a NOD scid gamma (NSG) mouse.

Para. E. The transplantable tumor line any one Paras. A-D, wherein thehFL-HCC cells are derived from a tumor removed from the liver, from thebiliary tree, from a subcutaneous tumor or from an intraperitoneal(ascites) tumor.

Para. F. The transplantable tumor line of any one of Paras A-E, whereinthe tumor line comprises hFL-HCC cells and mesenchymal cells of thenon-human animal.

Para. G. The transplantable tumor line of any one Paras. A-F, wherein atleast 50% of the hFL-HCC cells in the transplantable tumor are cancerstem cells.

Para. H. The transplantable tumor line of any one of Paras. A-G, wherethe mesenchymal cells are derived from the non-human animal.

Para. I. The transplantable tumor line of any one of Paras. A-H, whereinthe human FL-HCC cells express the fusion transcript DNAJB1-PRKACA.

Para. J. The transplantable tumor of any one of Paras. A-I, wherein thehFL-HCC cells substantially do not express HDAC9 or express a lowerlevel of HDAC9 as compared to a human non-FL-HCC cell control sample.

Para. K. The transplantable tumor line of any one of Paras. A-J, whereinthe hFL-HCC cells express one or more markers of endodermaltranscription factors selected from the group consisting of SOX9, SOX17,PDX1, FOXA1, and NGN3.

Para. L. The transplantable tumor line of any one of Paras. A-K, whereinthe hFL-HCC cells express one or more markers of pluripotency genesselected from the group consisting of OCT4, SOX2, NANOG, SALL4, KLF4,and KLF5.

Para. M. The transplantable tumor of any one of Paras. A-L, wherein thehFL-HCC cells express one or more markers of other stem cell genesselected from the group consisting of CD44, SALL4, TROP-2, BMI-1, sonichedgehog (SHH), LGR5, NCAM, and KRT20.

Para. N. The transplantable tumor line of any one of Paras. A-M, whereinthe hFL-HCC cells express one or more hepatic markers selected from thegroup consisting of CK8, CK18, CK19, DCLK1, HepPar-1, albumin,alpha-fetoprotein, and CD68.

Para. O. The transplantable tumor line of any one of Paras. A-N, whereinthe hFL-HCC cells express one or more pancreatic markers selected fromPDX1, PCSK1, NGN3, insulin, glucagon, amylase, and mucin (MUC).

Para. P. The transplantable tumor line of any one of Paras. A-O, whereinthe hFL-HCC cells express high levels of aryl hydrocarbon receptors(AHR).

Para. Q. The transplantable tumor line of any one of Paras. A-P, whereinthe hFL-HCC cells express biomarkers of malignancy such as AGR2 and/orhigh levels of extracellular matrix-degrading enzymes and/or aberrationsin the regulation of p53.

Para. R. The transplantable tumor line of any one of Paras. A-Q, whereinthe hFL-HCC cells have aberrant or lack of expression of one or morehistone deacetylase (HDAC) genes.

Para. S. A transplantable tumor line comprising human FL-HCC (hFL-HCC)cells and mesenchymal cells from a non-human host.

Para. T. The tumor line of Para. S, wherein the non-human host is animmunocompromised mouse.

Para. U. The tumor line of Para. S or T, wherein the non-human host is aNOD scid gamma mouse

Para. V. The tumor line of Para. S, which is a xenotransplanted,subcutaneous or intraperitoneal tumor.

Para. W. The tumor line of Para. S, wherein at least 30% of the hFL-HCCcells are cancer stem cells

Para. X. The tumor line of Para. S, wherein at least 50% of the hFL-HCCcells are cancer stem cells.

Para. Y. The tumor line of Para. S, wherein at least 65% of the hFL-HCCcells are cancer stem cells.

Para. Z. The tumor line of any one of Paras. S-Y, wherein the hFL-HCCcells express the fusion transcript DNAJB1-PRKACA.

Para. AA. The tumor line of any one of Paras. S-Z, wherein the hFL-HCCcells substantially do not express or express low levels of HDAC9 orexpress a lower level of HDAC9 as compared to a human non-FL-HCC cellcontrol sample.

Para. AB. The tumor line of any one of Paras. S-AA, wherein the hFL-HCCcells express one or more markers of endodermal transcription factorsselected from the group consisting of SOX9, SOX17, PDX1, FOXA1, andNGN3.

Para. AC. The tumor line of any one of Paras. S-AB, wherein the hFL-HCCcells express one or more markers of pluripotency genes selected fromthe group consisting of OCT4, SOX2, NANOG, SALL4, KLF4, and KLF5.

Para. AD. The tumor line of any one of Paras. S-AC, wherein the hFL-HCCcells express one or more markers of other stem cell genes selected fromthe group consisting of CD44, SALL4, TROP-2, BMI-1, sonic hedgehog(SHH), LGR5, NCAM, and KRT20.

Para. AE. The tumor line of any one of Paras. S-AD, wherein the humanFL-HCC cells express one or more hepatic markers selected from the groupconsisting of CK7, CK8, CK18, CK19, DCLK1, HepPar-1, albumin,alpha-fetoprotein, and CD68.

Para. AF. The tumor line of any one of Paras. S-AE, wherein the hFL-HCCcells express one or more pancreatic markers selected from the groupconsisting of PDX1, PCSK1, NGN3, insulin, glucagon, amylase, and mucin(MUC).

Para. AG. The tumor line of any one of Paras. S-AF, wherein the hFL-HCCcells express high levels of aryl hydrocarbon receptors (AHR).

Para. AH. The tumor line of any one of Paras. S-AG, wherein the hFL-HCCcells express biomarkers of malignancy such as AGR2 and/or high levelsof extracellular matrix-degrading enzymes.

Para. AI. The tumor line of any one of Paras. S-AH, wherein the humanFL-HCC cells have aberrant or lack of expression of one or more histonedeacetylase (HDAC) genes.

Para. AJ. A tissue sample obtained from the tumor line of any one ofParas. R-AI.

Para. AK. A cell culture comprising hFL-HCC cells in a serum-freemedium.

Para. AL. The cell culture of Para. AK, wherein the serum-free medium isKubota's Medium.

Para. AM. The cell culture of Para. AK or AL, wherein the serum-freemedium contains hyaluronans, HGF and/or VEGF.

Para. AN. The cell culture of any one of Paras. AK-AM, wherein at least51% of the cells in the cell culture are hFL-HCC cells.

Para. AO. The cell culture of any one of Paras. AK-AN, wherein at least50% of the hFL-HCC cells in the cell culture are cancer stem cells.

Para. AP. The cell culture of any one of Paras. AK-AO, wherein at leasta portion of the hFL-HCC cells are in aggregates of hFL-HCC cells.

Para. AQ. The cell culture of any one of Paras. AK-AP, wherein thehFL-HCC cells express fusion transcript DNAJB1-PRKACA.

Para. AR. The cell culture of any one of Paras. AK-AQ, wherein thehFL-HCC cells substantially do not express HDAC9 or express a lowerlevel of HDAC9 as compared to a human non-FL-HCC cell control sample.

Para. AS. The cell culture of any one of Paras. AK-AR, wherein thehFL-HCC cells express one or more endodermal transcription factorsselected from the group consisting of SOX9, SOX17, PDX1, and NGN3.

Para. AT. The cell culture of any one of Paras. AK-AS, wherein thehFL-HCC cells express one or more pluripotency genes selected from thegroup consisting of SOX2, NANOG, SALL4, OCT4, KLF4, and KLF5.

Para. AU. The cell culture of any one of Paras. AK-AT, wherein thehFL-HCC cells express one or more stem cell genes selected from TROP-2,SALL4, BMI-1, LGR5, sonic hedgehog (SHH), NCAM.

Para. AV. The cell culture of any one of Paras. AK-AU, wherein thehFL-HCC cells express one or more hepatic markers selected from thegroup consisting of CK7, CK8, CK18 CK19, HepPar-1, albumin,alpha-fetoprotein, LGR5, and CD68.

Para. AW. The cell culture of any one of Paras. AK-AV, wherein thehFL-HCC cells express one or more pancreatic markers selected from thegroup consisting of PDX1, NGN3, PCSK1, insulin, glucagon, amylase, andmucin (MUC).

Para. AX. A method for establishing a hFL-HCC tumor line comprising: (a)obtaining a human FL-HCC tumor from a patient with hFL-HCC; (b)preparing a tumor cell suspension from the hFL-HCC tumor; (c) culturingthe tumor cell suspension under restrictive conditions that select forcancer stem cells to obtain a population of culture-selected cancer stemcells; and (d) transplanting culture-selected cells into animmunocompromised, non-human animal

Para. AY. The method of Para. AX, in which the hFL-HCC tumor is obtainedas an ascites fluid or as a solid tumor from the subject.

Para. AZ. The method of Para. AX or AY, wherein the tumor cellsuspension from the hFL-HCC tumor are cultured on tissue culture plasticor on or in hyaluronans.

Para. BA. The method of any one of Paras. AX-AZ, wherein the tumor cellsuspension from the hFL-HCC tumor are cultured in serum-free Kubota'sMedium.

Para. BB. The method of any one of Paras. AX-BA, comprising at step (d)transplanting subcutaneously or intraperitoneally the culture-selectedcancer stem cells from the hFL-HCC cells into the immunocompromisednon-human animal.

Para. BC. The method of any one of Paras. AX-BB, comprising at step (d)transplanting about 10² to about 10⁷ culture-selected cancer stem cellsfrom the hFL-HCC tumor into the immunocompromised, non-human animal.

Para. BD. The method of any one of Paras. AX-BC, further comprisingafter step (d) monitoring the immunocompromised, non-human animal fortumor formation for about 2 to about 9 months.

Para. BE. A method for maintaining a hFL-HCC transplantable tumor linecomprising: (a) obtaining hFL-HCC cells from a xenografted tumor of amaintained in an immunocompromised non-human animal, (b) dispersing thehFL-HCC cells into a cell suspension by enzymatic and/or mechanicalmethods, and (c) transplanting dispersed hFL-HCC cells into a secondimmunocompromised, non-human animal.

Para. BF. The method of Para. BE, comprising culturing the hFL-HCC cellsin serum-free medium.

Para. BG. The method of Para. BF, wherein the serum-free medium isKubota's Medium.

Para. BH. The method of Para. BE, wherein the serum-free medium furthercontains hyaluronans, HGF and/or VEGF.

Para. BI. The method of any one of Paras. BE-BH comprising, at step (c)transplanting subcutaneously or intraperitoneally the hFL-HCC tumor intothe second immunocompromised, non-human animal.

Para. BJ. A method for culturing hFL-HCC cells comprising: (a)separating hFL-HCC cells of a xenografted tumor from non-human cells;(b) suspending the separated hFL-HCC cells in a serum-free medium, and(c) plating the hFL-HCC cells as monolayers onto or into a culturesubstratum to obtain plated hFL-HCC cells or allowing the cells to formfloating aggregates.

Para. BK. The method of Para. BJ, comprising at step (c) separatinghFL-HCC cells from non-human cells by magnetic immunoselection.

Para. BL. The method of Para. BJ, wherein the culture substratum is atissue culture plastic, a surface coated with a purified extracellularmatrix component or with an extract enriched in extracellular matrix, a3D hydrogel of a purified extracellular matrix component, or asuspension.

Para. BM. The method of Para. BL, wherein the purified extracellularmatrix component is selected from the group consisting of hyaluronan, acollagen, an adhesion molecule, and an extract enriched in extracellularmatrix.

Para. BN. The method of Para. BM, wherein the adhesion molecule islaminin.

Para. BO. The method of Para. BM, wherein the extract enriched inextracellular matrix is a matrix scaffold, a biomatrix scaffold, orMatrigel.

Para. BP. The method of Para. BJ, wherein the plated hFL-HCC cells arekept in suspension and allowed to form aggregates.

Para. BQ. A method for drug screening, comprising (a) introducing acandidate drug to cultured hFL-HCC cells that are in the form ofmonolayers, hydrogels, spheroids or organoids, and (b) monitoring theeffect of the candidate drug on the cultured hFL-HCC cells.

Para. BR. A method for drug testing, comprising (a) administering acandidate drug to a non-human animal carrying a transplanted hFL-HCCtumor and (b) monitoring the effect of the candidate drug on thexenotransplanted hFL-HCC tumor.

Para. BS. A method for suppressing the growth of hFL-HCC cells,comprising treating the hFL-HCC cells with a hedgehog signalinginhibitor, a histone deacetylase inhibitor, a protein kinase inhibitor,and/or an inhibitor of a gene overexpressed in hFL-HCC cells.

Para. BT. The method of Para. BS, wherein the hedgehog signaling pathwayinhibitor comprises GDC-0449.

Para. BU. The method of Para. BS, wherein the histone deacetylaseinhibitor comprises suberoylanilide hydroxamic acid (SAHA) and/orsuberic bis-hydroxamic acid (SBHA)

Para. BV. A method for treating hFL-HCC in a patient in need thereof,comprising administering to the patient an effective amount of ahedgehog signaling pathway inhibitor, a histone deacetylase inhibitor, aprotein kinase inhibitor, and/or an inhibitor of a gene overexpressed inhFL-HCC cells.

Para. BW. The method of Para. BV, wherein the hedgehog signaling pathwayinhibitor comprises GDC-0449

Para. BX. The method of Para. BX, wherein the histone deacetylaseinhibitor comprises SAHA and/or SBHA.

Para. BY. A method of determining whether a patient has fibrolamellarhepatocellular carcinoma (FL-HCC), comprising: (a) measuring geneexpression levels of at least one of C10orf128, CA12, CREB3L1, GALNTL6,IRF4, ITPRIP, KCNE4, NOVA1, OAT, PAK3, PCSK1, PHACTR2, RPS6KA2,SLC16A14, TMEM163, and TNRC6C; and (b) comparing the gene expressionprofile to one or more control samples.

Para. BZ. The method of Para. BY, wherein overexpression of C10orf128,CA12, CREB3L1, GALNTL6, IRF4, ITPRIP, KCNE4, NOVA1, OAT, PAK3, PCSK1,PHACTR2, RPS6KA2, SLC16A14, TMEM163 or TNRC6C relative to the controlsample is associated with presence of FL-HCC.

Para. CA. The method of Para. BZ, wherein overexpression of PCSK1, CA12,NOVA1, SLC16A14, TNRC6C, TMEM163, and RPS6KA2 relative to the controlsample is associated with presence of FL-HCC.

Para. CB. The method of Para. BZ, wherein overexpression of C10orf128,OAT, PAK3, PCSK1, PHACTR2, SLC16A14, TMEM163, and TNRC6C relative to thecontrol sample is associated with presence of FL-HCC.

Para. CC. The method of any one of Paras. BZ-CB, wherein the controlsample is selected from the tumor cells from hepatocellular carcinomas(HCCs), hepatoblastomas, cholangiocarcinomas (CCAs) and/or pancreaticcancers or selected from normal cells consisting of biliary tree stemcells, hepatic stem cells, hepatoblasts, pancreatic stem cells, hepaticor pancreatic committed progenitors, and normal mature hepatic orpancreatic cells.

Para. CD. A method of treating a patient determined to have hFL-HCC byadministering to the patient an effective amount of at least onetherapeutic that decreases expression of at least one of C10orf128,CA12, CREB3L1, GALNTL6, IRF4, ITPRIP, KCNE4, NOVA1, OAT, PAK3, PCSK1,PHACTR2, RPS6KA2, SLC16A14, TMEM163, or TNRC6C.

Para. CE. The method of Para. CD, wherein the at least one therapeuticis selected from the group consisting of a small molecule, RNAinterference, and a locked nucleic acid (LNA).

Para. CF. A method of treating a patient determined to have hFL-HCC byadministering to the patient an effective amount of an immunotherapy.

Para. CG. A method of treating a patient determined to have hFL-HCC byadministering to the patient an effective amount of at least onetherapeutic that regulates PRKACA or SRC network hubs.

Para. CH. A method of treating a patient determined to have hFL-HCC byadministering to the patient an effective amount of at least onetherapeutic that regulates substrate targets of the kinase PRKACA(Protein kinase A catalytic subunit alpha).

Para. CI. An isolated hFL-HCC cell wherein the hFL-HCC cell expresses amarker selected from the group consisting of C10orf128, CA12, CREB3L1,GALNTL6, IRF4, ITPRIP, KCNE4, NOVA1, OAT, PAK3, PCSK1, PHACTR2, RPS6KA2,SLC16A14, TMEM163, and TNRC6C.

Para. CJ. A population of isolated hFL-HCC cells of Para. CI.

Para. CK. A composition comprising an isolated hFL-HCC cell of Para. CIor CJ and a carrier.

Para. CL. The composition of any one of Paras. CI-CK, wherein thehFL-HCC cells are obtained from ascites fluid or a solid tumor.

Para. CM. The composition of any one of Paras. CI-CL, wherein thehFL-HCC cells are cultured on tissue culture plastic or on or inhyaluronans.

Para. CN. The composition of any one of Paras. CI-CM, wherein thehFL-HCC cells are cultured in cells in serum-free medium.

Para. CO. The composition of any one of Paras. CI-CN, wherein theserum-free medium is Kubota's Medium.

Para. CP. The composition of Paras. CI-CO, wherein the serum-free mediumfurther contains hyaluronans, HGF and/or VEGF.

Para. CQ. The composition of any one of Paras. CI-CP, further comprisingpurified extracellular matrix component.

Para. CR. The composition of Para. CQ, wherein the purifiedextracellular matrix component is selected from the group consisting ofhyaluronan, a collagen, an adhesion molecule, and an extract enriched inextracellular matrix.

Para. CS. The composition of Para. CR, wherein the adhesion molecule islaminin.

Para. CT. The composition of Para. CR, wherein the extract enriched inextracellular matrix is a matrix scaffold, a biomatrix scaffold, orMatrigel.

Para. CU. A transplantable tumor cell line comprising humanfibrolamellar hepatocellular carcinoma (hFL-HCC) cells, which can bemaintained in a non-human animal.

What is claimed is:
 1. A transplantable tumor line of human fibrolamellar hepatocellular carcinoma (hFL-HCC) cells maintained in a non-human animal.
 2. The transplantable tumor line of claim 1, wherein the non-human animal is a NOD scid gamma (NSG) mouse.
 3. The transplantable tumor line of claim 1, wherein the hFL-HCC cells are derived from a tumor removed from the liver, from the biliary tree, from a subcutaneous tumor or from an intraperitoneal (ascites) tumor.
 4. The transplantable tumor line of claim 1, wherein the tumor line comprises hFL-HCC cells and mesenchymal cells of the non-human animal.
 5. The transplantable tumor line of claim 1, wherein at least 50% of the hFL-HCC cells in the transplantable tumor are cancer stem cells.
 6. The transplantable tumor line of claim 1, wherein the hFL-HCC cells express the fusion transcript DNAJB1-PRKACA.
 7. The transplantable tumor line of claim 1, wherein the hFL-HCC cells overexpress at least one C10orf128, CA12, CREB3L1, GALNTL6, IRF4, ITPRIP, KCNE4, NOVA1, OAT, PAK3, PCSK1, PHACTR2, RPS6KA2, SLC16A14, TMEM163, or TNRC6C relative to a control sample.
 8. The transplantable tumor line of claim 7, wherein the control sample is selected from the group consisting of hepatocellular carcinomas (HCCs), hepatoblastomas, cholangiocarcinomas (CCAs), pancreatic cancer, biliary tree stem cells, hepatic stem cells, hepatoblasts, pancreatic stem cells, hepatic, pancreatic committed progenitors, and normal mature hepatic or pancreatic cells.
 9. A tissue sample obtained from the tumor line of claim
 1. 10. A population of hFL-HCC cells isolated from the tumor line of claim
 1. 11. The population of claim 10, wherein the hFL-HCC cells are cultured on tissue culture plastic or on or in hyaluronans.
 12. The composition of claim 11, wherein the hFL-HCC cells are cultured in cells in serum-free medium.
 13. The composition of claim 12, wherein the serum-free medium is Kubota's Medium.
 14. The composition of claim 12, wherein the serum-free medium further contains hyaluronans, HGF and/or VEGF.
 15. A method of determining whether a patient has fibrolamellar hepatocellular carcinoma (FL-HCC), comprising: (a) measuring gene expression levels of at least one of C10orf128, CA12, CREB3L1, GALNTL6, IRF4, ITPRIP, KCNE4, NOVA1, OAT, PAK3, PCSK1, PHACTR2, RPS6KA2, SLC16A14, TMEM163, and TNRC6C; and (b) comparing the gene expression profile to one or more control samples.
 16. The method of claim 15, wherein overexpression of C10orf128, CA12, CREB3L1, GALNTL6, IRF4, ITPRIP, KCNE4, NOVA1, OAT, PAK3, PCSK1, PHACTR2, RPS6KA2, SLC16A14, TMEM163 or TNRC6C relative to the control sample is associated with presence of FL-HCC.
 17. The method of claim 16, wherein overexpression of PCSK1, CA12, NOVA1, SLC16A14, TNRC6C, TMEM163, and RPS6KA2 relative to the control sample is associated with presence of FL-HCC.
 18. The method of claim 16, wherein overexpression of C10orf128, OAT, PAK3, PCSK1, PHACTR2, SLC16A14, TMEM163, and TNRC6C relative to the control sample is associated with presence of FL-HCC.
 19. The method of claim 15, wherein the control sample is selected from the tumor cells from hepatocellular carcinomas (HCCs), hepatoblastomas, cholangiocarcinomas (CCAs) and/or pancreatic cancers or selected from normal cells consisting of biliary tree stem cells, hepatic stem cells, hepatoblasts, pancreatic stem cells, hepatic or pancreatic committed progenitors, and normal mature hepatic or pancreatic cells.
 20. A method of treating a patient determined to have hFL-HCC by administering to the patient an effective amount of at least one therapeutic that decreases expression of at least one of C10orf128, CA12, CREB3L1, GALNTL6, IRF4, ITPRIP, KCNE4, NOVA1, OAT, PAK3, PCSK1, PHACTR2, RPS6KA2, SLC16A14, TMEM163, or TNRC6C.
 21. The method of claim 20, wherein the at least one therapeutic is selected from the group consisting of a small molecule, RNA interference, a locked nucleic acid (LNA), an immunotherapy, a hedgehog signaling inhibitor, a histone deacetylase inhibitor, a protein kinase inhibitor, and a regulator of substrate targets of PRKACA.
 22. A method for drug screening, comprising (a) introducing a candidate drug to cultured hFL-HCC cells that are in the form of monolayers, hydrogels, spheroids or organoids, and (b) monitoring the effect of the candidate drug on the cultured hFL-HCC cells.
 23. A transplantable tumor cell line comprising human fibrolamellar hepatocellular carcinoma (hFL-HCC) cells, which can be maintained in a non-human animal. 